Particle switching systems and methods using acoustic radiation pressure

ABSTRACT

The present invention comprises methods and systems that use acoustic radiation pressure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of PatentCooperation Treaty Application Serial No. PCT/US08/59181, entitled“Methods and Devices for Enhanced Analysis of Field Focused Cells andParticles” filed Apr. 2, 2008, which claims priority of U.S. ProvisionalPatent Application Ser. No. 60/909,704, entitled “Methods and Devicesfor Enhanced Analysis of Field Focused Cells and Particle”, filed Apr.2, 2007, and of U.S. Provisional Patent Application Ser. No. 61/026,082,entitled “Applications and Methods for Field-Based Manipulation of Cellsand Particles Through Flow Lines of Heterogenous Media”, filed Feb. 4,2008, and the specifications and claims thereof are incorporated hereinby reference.

This application also claims priority to and the benefit of the filingof U.S. Provisional Patent Application Ser. No. 61/026,082, entitled“Applications and Methods for Field-Based Manipulation of Cells andParticles Through Flow Lines of Heterogenous Media”, filed Feb. 4, 2008,and the specification thereof is incorporated herein by reference.

This application is also related to the following applications filed oneven date herewith: “Medium Switching Systems and Methods Using AcousticRadiation Pressure,” Ser. No. 12/239,390; “Particle Analyzing Systemsand Methods Using Acoustic Radiation Pressure” Ser. No. 12/239,453;“Particle Imaging Systems and Methods Using Acoustic RadiationPressure,” Ser. No. 12/239,467; and “Particle Fusing Systems and MethodsUsing Acoustic Radiation Pressure,” Ser. No. 12/239,483; “Kits forSystems and Methods Using Acoustic Radiation Pressure,” Ser. No.12/239,501; “Particle Quantifying Systems and Methods Using AcousticRadiation Pressure,” Ser. No. 12/239,513 and the specifications andclaims thereof are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

Embodiments of the present invention relate to systems using acousticradiation pressure.

2. Background

Note that the following discussion refers to a number of publications byauthor(s) and year of publication, and that due to recent publicationdates certain publications are not to be considered as prior artvis-a-vis the present invention. Discussion of such publications hereinis given for more complete background and is not to be construed as anadmission that such publications are prior art for patentabilitydetermination purposes.

Flow cytometry is a powerful tool used for analysis of particles andcells in a myriad of applications primarily in bioscience research andmedicine. The analytical strength of the technique lies in its abilityto parade single particles (including bioparticles such as cells,bacteria and viruses) through the focused spot of light sources,typically a laser or lasers, in rapid succession, at rates up tothousands of particles per second. The high photon flux at this focalspot produces scatter of light by a particle and or emission of lightfrom the particle or labels attached to the particle that can becollected and analyzed. This gives the user a wealth of informationabout individual particles that can be quickly parleyed into statisticalinformation about populations of particles or cells.

In traditional flow cytometry, particles are flowed through the focusedinterrogation point where a laser directs a laser beam to a focusedpoint that includes the core diameter within the channel. The samplefluid containing particles is focused to a very small core diameter ofaround 10-50 microns by flowing sheath fluid around the sample stream ata very high volumetric rate on the order of 100-1000 times thevolumetric rate of the sample. This results in very fast linearvelocities for the focused particles on the order of meters per second.This in turn means that each particle spends a very limited time in theexcitation spot, often only 1-10 microseconds. Further, once theparticle passes the interrogation point the particle cannot beredirected to the interrogation point again because the linear flowvelocity cannot be reversed. Further, a particle cannot be held at theinterrogation point for a user defined period of time for furtherinterrogation because focusing is lost without the flow of thehydrodynamic sheath fluid. Because of the very high photon flux at theexcitation point, flow cytometry is still a very sensitive technique,but this fast transit time limits the sensitivity and resolution thatcan be achieved. Often, greater laser power is used to increase thephoton flux in an effort to extract more signal but this approach islimiting in that too much light can often photobleach (or excite tonon-radiative states) the fluorophores being used to generate the signaland can increase background Rayleigh scatter, Raman scatter andfluorescence.

Acoustic cytometers, using relatively large dimension flow channels,concentrate particles from the entire volume of the channel to a smallacoustic trap in the center of the channel and can therefore offer bothcontrollable flow and high particle analysis rates without resorting tohighly concentrated samples.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the present invention comprises a method foracoustically reorienting a fluid in a channel. This method preferablyincludes introducing into a channel a first fluid wherein the firstfluid has a first acoustic contrast relative to a second fluid,introducing into the channel the second fluid wherein the second fluidhas a second acoustic contrast that is different from the first acousticcontrast, applying acoustic radiation pressure to the channel, andacoustically reorienting the second fluid based upon the acousticcontrast of the second fluid. The method can acoustically reorient thefirst fluid and the second fluid relative to particles. The first fluidand/or the second fluid preferably move in laminar flow streams. Themethod can also further comprise assaying the particles or fluids toproduce an assay. An assay is preferably produced by the method whereinthe second fluid is a biological fluid selected from the groupconsisting of cell culture medium, serum, blood, bone marrow, semen,vaginal fluid, urine, spinal fluid, saliva, sputum, bile, peritonealfluid, amniotic fluid, and aspirate from hollow organs, cysts andtissue. One of the fluids of the method can comprise a reagent. Thereagent can be selected from a) an antibody or aptamer specific for aparticle antigen; b) a ligand specific for a particle receptor; c) anenzyme specific for a particle substrate; d) a nucleic acid stainspecific for particle nucleic acid; e) an antigen specific for aparticle antibody, f) an analyte specific for a particle target; g) asecondary reagent specific for one or more of a-f; and h) anycombination thereof. The method can optionally include passing aparticle through a zone for collection of luminescence and collectingchemi, bio or electro luminescence from the particle. The particles canbe focused with a radial acoustic field. The collecting of luminescencecan occur between excitation pulses from a light source.

Another embodiment of the present invention comprises a method foracoustically manipulating a particle using radial acoustic focusing.This embodiment preferably comprises introducing into a channel a firstfluid having a first acoustic contrast with a population of particlessuspended therein, introducing into the channel a second fluid having asecond acoustic contrast that is greater than or equal to the acousticcontrast of the first fluid, applying a radial acoustic radiationpressure to the channel, and acoustically focusing at least a portion ofthe population of particles from the first fluid to the second fluid.The first fluid and/or the second fluid preferably move in laminar flowstreams. A portion of the population of particles can be acousticallyfocused relative to the first acoustic contrast and the second acousticcontrast. In this embodiment a subset of particles may be more than onesize and a first particle size can be acoustically focused more quicklyinto the second fluid than a subset of particles having a secondparticle size. Particles useful as standards in flow cytometry havingbeen acoustically separated by size and having an improved coefficientof variation after acoustic separation as compared to the startingpopulation of particles wherein the particles are produced by the methodof this embodiment. The first fluid of this embodiment can comprise areagent that specifically binds at least a subset of the portion of thepopulation of particles. The method can also further comprisenon-dilutive sorting of the portion of the population of particles inthe second fluid. The first and/or second fluid of this embodiment cancomprise a reagent that reacts with at least some of the particles fromthe portion of the population of particles in the second fluid. Thereagent can be selected from a) antibodies or aptamers specific forparticle antigens; b) ligands specific for particle receptors; c) enzymespecific for particle substrate; d) stains specific for particle nucleicacid; e) an antigen specific for a particle antibody, f) an analytespecific for a particle target; g) a secondary reagent specific for oneor more of a-f; and h) any combination thereof.

The method described above may also have the second fluid located at thecenter of the channel. In addition, the particles can be in the firstfluid or the second fluid to a particle analyzer, e.g. a flow cytometerthat is in line with the channel. Acoustically focusing in thisembodiment can comprise acoustically focusing a subset of the populationof particles with greater contrast to the second stream for collectionand excluding a subset of the population of particles with a lessercontrast from collection. The population of particles in this embodimentmay comprise an array of beads having a target specific for a pre-boundfluorescent analyte of interest and wherein the second fluid issuspected of containing non fluorescent analyte that is capable ofbinding specifically to its target on a bead from the array of bead andfurther comprising displacing the pre-bound fluorescent analyte with anon-fluorescent analyte when the bead is acoustically focused in thesecond fluid and analyzing the array of beads for fluorescence.

The method of the embodiment above can further comprise acousticallyfocusing the cell into a reagent loaded second fluid and providing tothe cell an electric field that permeates the cell membrane to permitthe reagent to cross into the permeated cell.

The method of the embodiment above may also comprise a reagent thatbinds to at least a portion of the subset of the population of particlesto form a particle-reagent complex having an acoustic contrast differentthan the acoustic contrast of the portion of the population of particlesnot complexed with reagent. The particle-reagent complex is acousticallyfocused away from at least a portion of the population of particles notcomplexed with reagent.

The population of particles can be cultured cells, the first fluid canbe cell growth medium in which the cells are grown and the second fluidcan be new cell growth medium.

The method can further comprise capturing a particle of interest fromthe population of particles with a negative contrast particle andforcing the particle of interest and the negative contrast particletoward a wall of the channel away from a center of the channel.

The method can alternatively comprise producing an acoustic node outsidethe channel wherein the reorienting of the second fluid with the atleast a portion of the particles therein is to the top surface of thechannel near the acoustic node.

The method can additionally comprise introducing a calcium sensitivereagent into a cell, moving the cell through a channel, acousticallyfocusing the cell within the channel, exposing the cell to a reagentthat may or may not induce a cellular calcium response, passing the cellthrough an interrogation site, and collecting a signal to determinecalcium concentration in the cell. In this method, the cell ispreferably acoustically washed and/or diluted prior to collection. Thismethod can further comprise adjusting a flow rate to achieve a desiredtime of collection after the exposure to the reagent that may or may notinduce a calcium response.

Yet another embodiment of the present invention comprises a method forimaging acoustically manipulated particles in an acoustic flowcytometer. This method preferably comprises introducing a fluidcontaining a population of particles therein to a flow cell, applyingacoustic radiation pressure to the flow cell, acoustically focusing thepopulation of particles within the flow cell to concentrate thepopulation of particles, aligning some of the concentrated population ofparticles in the flow cell, interrogating some of the aligned populationof particles at an interrogation site to obtain an optical signal fromsome of the population of particles to yield population statisticaldata, and imaging at least one of the population of particles to producea high content image representative of the population of particles. Themethod can further comprise the step of correlating the populationstatistical data with the high content image to produce improved datacontent. The transit time of the population of particles may also beslowed during the interrogating step.

One embodiment of the present invention is a method for optimizingparticle throughput in a particle analyzer for a user defined transittime with a given concentration of particles. This method preferablycomprises the steps of determining optimal concentration of particles toachieve a user defined coincidence rate, adjusting sample concentrationto achieve the coincidence rate, acoustically focusing particles in theparticle analyzer, adjusting flow rate to achieve desired user definedtransit time of the particles, and analyzing the particles at aninterrogation point. The particle analyzer can be a flow cytometer orcell impedance analyzer.

Another embodiment of the present invention comprises a method of fusingan antibody producing cell with an immortal cell to produce a hybridomacell. This embodiment preferably comprises acoustically focusing in afirst channel having a first acoustic field an antibody producing cellcapable of fusing with an immortal cell, acoustically focusing theimmortal cell in a second channel having a second acoustic field,flowing the acoustically focused antibody producing cell and theacoustically focused immortal cell to a third channel having a thirdacoustic field that acoustically focuses the antibody cell and theimmortal cell into close enough proximity to permit the antibody celland the immortal cell to fuse, and fusing the antibody cell and theimmortal cell together by a chemical or electrical means to form ahybridoma cell. A hybridoma cell line is preferably created by themethod.

Yet another embodiment of the present invention comprises a method ofanalyzing particles with a particle analyzer. This method preferablycomprises the steps of acoustically focusing particles to theapproximate center of a channel having an electrolytic fluid therein,flowing the particles through a pore of the channel separating twoelectrodes between through which an electric current flows, anddetecting the signal wherein the particles displace their own volume ofelectrolyte momentarily increasing the impedance of the pore to producethe signal. The electrolytic fluid of this embodiment is preferably ofan engineered conductivity. In addition, a fluid for use in the methodpreferably has an engineered conductivity. The particles in this methodare preferably blood cells.

A further embodiment of the present invention comprises a method ofacoustically focusing particles in a plane. This embodiment preferablycomprises transiting a fluid containing particles therein through achannel at a flow rate, adjusting the flow rate for a desired transittime through an excitation source, optically exciting the particles withthe excitation source, detecting an optical signal from the particles,and analyzing the optical signal. This embodiment can further comprisedisposing a substantially acoustically transparent gas-contactingmembrane at a top surface of the flow channel wherein a pressure node islocated outside of the channel at a gas interface.

Another embodiment of the present invention comprises a kit foracoustically focusing at least one particle. This kit preferablyincludes a container means having a first fluid, the container meanshaving a population of particles therein wherein the population ofparticles are engineered for an acoustic radiation pressure apparatus,wherein the particles having an acoustic contrast greater than the firstfluid and the first fluid has an acoustic contrast that is greater thanwater and wherein the first fluid comprises one or more engineeredpopulation of particles. In this embodiment, the first fluid preferablycomprises a heavy salt. The heavy salt is selected from the groupconsisting of potassium bromide and cesium chloride. Alternatively, thefirst fluid can comprise an iodinated compound. The iodinated compoundis selected from the group consisting of metrizamide, Nycodenz®,diatrizoate, and iodixanol. The first fluid can also comprisenanoparticles or sucrose, polysucrose, polydextran, and glycerol. Thepopulation of particles is preferably a material selected frompolystyrene, acrylic, iron oxides, silica or any combination thereof.The acoustic contrast of the population of particles is preferablygreater than a target acoustic contrast. The population of particles cancomprise a first subset of the population and second subset of thepopulation wherein the first subset of the population is different thanthe second subset of the population. The first subset of the populationpreferably has a probe specific for a first analyte and the secondsubset of the population preferably has a probe specific for a secondanalyte. The first analyte of this embodiment of preferably differentfrom the second analyte. The first population of particles can labeledwith a first signaling molecule having a first lifetime and the secondpopulation of particles can labeled with a second signaling moleculehaving a second lifetime.

Still another embodiment of the present invention comprises a kit foracoustic focusing. This kit preferably includes a container means havinga first fluid, and a container means having a reagent for labeling aparticle, wherein the particle has an acoustic contrast greater than thefirst fluid, the first fluid has an acoustic contrast that is greaterthan water and wherein the first fluid comprises one or more engineeredparticles engineered specifically for an acoustic radiation pressureapparatus. The particle of this kit preferably comprises a materialselected from polystyrene, acrylic, iron oxides, silica or anycombination thereof. The particle acoustic contrast is preferablygreater than the reagent acoustic contrast. Alternatively, the particlecan be a white blood cell, and the reagent is an antibody or fabfragment that specifically binds to a surface receptor on the whiteblood cell. The first fluid comprises heavy salt. The heavy salt isselected from the group consisting of potassium bromide and cesiumchloride. The first fluid can alternatively comprise an iodinatedcompound. The iodinated compound is selected from the group consistingof metrizamide, Nycodenz®, diatrizoate, and iodixanol. The first fluidcan alternatively comprise nanoparticles or one or more of the followingsucrose, polysucrose, polydextran, and glycerol.

One embodiment of the present invention is a kit for reagents for use inan acoustic apparatus with two streams. This kit preferably includes afirst fluid for use in a first fluid stream of the acoustic apparatus, asecond fluid and a particle wherein the second fluid is used in a secondstream, and instructions for acoustically focusing the particle in anacoustic flow cytometer. The first fluid preferably has an acousticcontrast that is less than the acoustic contrast of the second fluid andless than the acoustic contrast of the particles in the first fluid. Thesecond fluid preferably has an acoustic contrast that is greater thanthe acoustic contrast of the first fluid and less than the acousticcontrast of the particles. The particle preferably binds a target. Theparticles preferably comprise a material selected from polystyrene,acrylic, iron oxides, silica or any combination thereof. The acousticcontrast of the particle is preferably greater than target acousticcontrast. The first fluid in this kit can be a wash fluid or a lysisfluid.

Another kit of the present invention is a kit for reagents for use in anacoustic apparatus with two streams. This kit preferably comprises afirst fluid, a second fluid and a reagent for labeling a particle. Thefirst fluid is preferably used in a first stream, and the second fluidis preferably used in a second stream. The first fluid preferably has anacoustic contrast that is less than the second fluid. The second fluidhas an acoustic contrast that is greater than the first fluid. Theparticles in this kit can be a white blood cell. The first fluid can bea wash fluid or a lysis fluid.

Another embodiment of the present invention comprises a method foranalyzing a particle in a long transit time flow cytometer. This methodpreferably comprises introducing a particle having a signaling moleculeassociated therewith to a particle analyzer having a laser interrogationlight source, interrogating the particle having the signaling moleculeassociated therewith repeatedly with the laser interrogation lightsource having interrogation light source bursts of about 0.1 ns-100 nswherein the burst occurs at a rate of about 0.01 MHz-2 Mhz to saturateor nearly saturate excitation of the signaling molecule, and collectingan optical signal from the signaling molecule and analyzing data toobtain information about the particle wherein the rest times are atleast 25% of the lifetime for a non-radiative decay rate.

Yet another embodiment of the present invention comprises a method ofmeasuring multiple analytes in a sample using an acoustic flowcytometer. This embodiment preferably comprises introducing a samplesuspected of containing one or more targets of interest into theacoustic flow cytometer, measuring the presence of the one or moretargets of interest with a first probe and a second probe wherein thefirst probe has a signaling molecule with a long optical lifetimeemission spectra and the second probe has a signaling molecule with ashorter optical lifetime emission spectra relative to the first probewherein the first probe and the second probe have a differentspecificity and wherein the signaling molecule with the long opticallifetime emission spectra and the signaling molecule with the shorteroptical lifetime emission spectra have overlapping emission spectra andwherein the one or more targets of interest are measured in an acousticflow cytometer, exciting the signaling molecule with a long opticallifetime and the signaling molecule with the shorter optical lifetimewith a pulsed light source, measuring the signal of both the long andshort lifetime signaling molecules and the signal of the long lifetimeprobe after the short lifetime probe signal has decayed, and calculatingthe contribution of each signaling molecule based on the combined signalof both molecules, the signal of the long lifetime molecule after thesignal from the short lifetime molecule has decayed and the knownlifetime curves of the signaling molecule.

The embodiment above can further comprise pooling an array of particlesubsets, each subset having a predetermined amount of at least one lightabsorbing dye and a reactant specific for the analyte, exposing thearray of particles to the sample containing the analyte, forming ananalyte-particle complex, passing the analyte-particle complex throughan interrogation site, determining the identity of each particle basedon its axial light loss absorbance measurement, and determining thepresence, quantity and identity of the analyte bound to the particlebased on data specific to the formation of the analyte-particle complex.This method can also optionally comprise reacting at least oneadditional reagent to the sample prior to passage through theinterrogation site and indicating the presence of the particle-analytecomplex. The interrogation site of this embodiment preferably comprisesat least one light source. The light source is preferably pulsed ormodulated. The particle array preferably has an additional fluorescentlabel that is used as a reference for quantification of the analyte.

One embodiment of the present invention comprises an acoustic flowcytometer capable of measuring axial light loss. This acoustic flowcytometer preferably comprises a channel having an inlet for accepting afluid sample stream of the analytes, an acoustic signal producingtransducer coupled to the channel wherein the acoustic signal producingtransducer produces an acoustic signal to the channel to induce withinthe channel acoustic radiation pressure capable of inducing an outerboundary surface displacement to concentrate the analytes within thefluid sample stream, optical equipment for analyzing the analyteswherein the optical equipment comprises a light source or light sourceswith one or more wavelengths for analyzing the analytes when theanalytes pass through the one or more wavelengths, and a linear arraydetector(s) to detect absorbance from the analytes to determinecharacteristics of the analytes.

Another embodiment of the present invention comprises a method of fusingparticle populations. This method preferably comprises the steps ofacoustically focusing in a first flow channel with a first acousticfield a first particle population capable of fusing with a secondparticle population, acoustically focusing the second particlepopulation in a second flow channel with a second acoustic field,flowing the acoustically focused first particle population and secondparticle population to a third flow channel with a third acoustic fieldthat acoustically focuses the first particle population and the secondparticle population into close enough proximity to permit the particlesto fuse, and fusing the first particle population and the secondparticle population by a physical, chemical or electrical means. Thismethod preferably produces a fused particle.

Yet another embodiment of the present invention comprises a method forseparating magnetic particles or magnetically stained cells. This methodpreferably comprises moving particles or cells with small enoughmagnetic susceptibility to avoid magnetic aggregation in the magneticfield of separation into a separation channel where they are drawn to asurface(s) in a region of gradient magnetic fields, magnetically movingthe particles to a surface(s) where they continue to move in thedirection of flow due to hydrodynamic forces, magnetically moving theparticles through at least one additional laminar stream of fluid beforereaching the surface(s), and moving the particles along the surface(s)in the magnetic field to a location where they can be collected. Thesurface of this method is preferably a magnetic wire or magneticallysusceptible feature spanning the height of the flow channel. A methodfor analyzing magnetic particles or magnetically stained cells in aparticle analyzer or the like preferably comprises flowing particles orcells with small enough magnetic susceptibility to avoid magneticaggregation in a magnetic focusing field, into a channel where they areexposed to gradient fields and moving particles along the surface in themagnetic field such that they are focused into flow lines that can beanalyzed downstream using optical detectors. The surface(s) of thisembodiment are preferably magnetic or magnetically susceptible taperedfeatures(s) that terminate prior to the analysis point. The particlesare preferably magnetically moved through at least one additionallaminar stream of fluid before reaching said surface.

One embodiment of the present invention is a method for fractionatingparticles. This embodiment preferably includes providing a fluid havingparticles therein, applying acoustic radiation pressure to the fluid,focusing the particles within the fluid into a single file line, movingthe particles in a flow rate, applying acoustic radiation pressure tothe fluid for a second time, focusing the particles based on size andacoustic contrast, producing at least two fluid fractions of theparticles, and collecting at least one of the fractions. The method mayfurther include adjusting the flow rate and/or the first or secondacoustic radiation pressure such that particles with different physicalproperties are diverted to different fluid fractions. The particles ofthis embodiment are preferably focused into the single file line usingan acoustic standing wave field. The acoustic standing wave field ispreferably generated using a radial focusing device. The fluid of thisembodiment is preferably drawn away from the sample prior to acousticfractionation in order to steer the particles to a different part of theacoustic fractionating field. Particles in flow cytometry having beenacoustically separated by size using this method are useful and have animproved coefficient of variation after acoustic separation as comparedto the starting population. The coefficient of variation can be improvedby >40% or even >80%.

A further embodiment of the present invention comprises a method forquantifying the amount of analyte bound to a particle in an acousticparticle analyzer. This method preferably comprises binding a particlehaving a known amount of calibration dye and a specificity for ananalyte to the analyte having a lifetime related signal to form ananalyte-particle complex, passing the analyte-particle complex throughan interrogation light source, measuring the signal related to thebinding event in an interrogation site, measuring an overlapping signalfrom the calibration dye, and calculating the amount of analyte presentby comparing the analyte related signal to the signal from thecalibration dye. The interrogation light source of this embodiment ifpreferably pulsed. The analyte having the short lifetime related signalcomprises a ligand with a label associated therewith wherein the ligandbinds with specificity to the analyte such that the particle and analyteand labeled ligand form a complex. The calibration dye preferably has along lifetime and the lifetime related signal of the analyte is a shortlifetime signal. Or the calibration dye can have a short lifetime andthe lifetime related signal of the analyte is a long lifetime signal.

Still another embodiment of the present invention is a method foridentifying and quantifying multiple analytes using coded beads from apooled population of beads and fluorescence lifetime in a particleanalyzer. This method preferably includes pooling an array of particlesubsets, each subset having a predetermined amount of at least one shortlifetime fluorescent label and at least one long lifetime fluorescentlabel and a reactant specific for an analyte, exposing the array ofparticles to a sample such that the analytes within the sample form ananalyte-particle complex, passing the array of particles through aninterrogation light source, determining the identity of each particlebased on its lifetime curve, and determining the presence, quantity andidentity of the analyte bound to the particle based on data specific tothe formation of the analyte-particle complex. This method can furthercomprise reacting at least one additional reagent to the sample prior topassing the array of particles through the interrogation light sourceand indicating the presence of the particle-analyte complex. This methodcan optionally comprise measuring the lifetime curve of each particleusing a single optical detector. The particle analyzer of thisembodiment is preferably an acoustic cytometer. The particle arraypreferably comprises an additional fluorescent label that is used as areference quantification of the analyte.

Another embodiment of the present invention includes a method forincreasing the dynamic range of measurements in an acoustic particleanalyzer. This method comprises passing particles capable of scatteringor emitting an optical signal through an interrogation site,interrogating each particle with an intensity modulated excitation lightsource for producing the optical signal comprising scatter and emissionsfrom the particles, collecting the optical signal, quantifying thestrongest scatter and emissions signals from a low excitation levelproduced by the intensity modulated excitation light source, andquantifying the weakest scatter and emissions signals from a highexcitation level produced by the intensity modulated excitation lightsource. The particle analyzer of this method is preferably an acousticcytometer. The modulated excitation light source is preferably pulsed.

Yet another embodiment of the present invention is a method forincreasing dynamic range measurement of a fluorescent bead having atleast two fluorescent color labels in known quantities in a particleanalyzer. This embodiment preferably includes the steps of passing aparticle through an interrogation site, simultaneously measuring a firstsignal of a first detector sensitive to one wavelength band of light anda second signal of a second detector sensitive to a different wavelengthband of light, and determining the ratio of each label to the otherlabel based on the ratio signal rise time in the first detector relativeto the second detector. The particle analyzer of this embodiment ispreferably an acoustic cytometer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is an illustration of field focused particles according to oneembodiment of the present invention.

FIGS. 2A and 2B illustrate a single line acoustic focusing deviceaccording to one embodiment of the present invention.

FIG. 3 illustrates a schematic of an acoustically driven flow cellfocusing particles to the center of a flowing liquid stream acrosslaminar flow lines according to one embodiment of the present invention.

FIGS. 4A and B illustrate one embodiment of an acoustically driven flowcell with two laminar flow streams in contact.

FIGS. 5A-5C illustrate the separation of micron sized polystyrenefluorescent orange/red particles from a background of nanometer sizedgreen particles in a homogeneous fluid according to one embodiment ofthe present invention. FIG. 5D illustrates a clean core streamintroduced alongside a coaxial stream containing fluorescent backgroundaccording to an embodiment of the present invention.

FIGS. 6A-6C illustrate particle separation across laminar flowboundaries for particles of different size according to one embodimentof the present invention.

FIGS. 7A, 7B, and 7C illustrate multiple embodiments of analysis in aflow cytometer like configuration for particles.

FIG. 8 illustrates a schematic of an acoustically focused flow cell incombination with an acoustic flow cytometer according to one embodimentof the present invention.

FIG. 9 illustrates a flow diagram according to one embodiment of thepresent invention.

FIG. 10 illustrates the flow diagram in FIG. 9 modified to includein-line laminar washing according to one embodiment of the presentinvention.

FIG. 11 illustrates field focusing of laminar wash fluid according toembodiments of the present invention.

FIG. 12 illustrates a schematic of parallel fluid switching deviceaccording to one embodiment of the present invention.

FIGS. 13A and 13B are schematics for stream switching of unlysed wholeblood according to one embodiment of the present invention.

FIG. 14 is a schematic of an acoustic stream switching particleimpedance analyzer according to one embodiment of the present invention.

FIG. 15 is a schematic example of separation of negative contrastcarrier particles from a core of blood sample according to oneembodiment of the present invention.

FIG. 16 illustrates a schematic example of multi-plexed competitiveimmunoassaying in an acoustic wash system according to one embodiment ofthe present invention.

FIG. 17 illustrates a flow chart for high throughput/high contentscreening using acoustic fluid switching according to one embodiment ofthe present invention.

FIG. 18 illustrates a schematic example of a two chamberculturing/harvesting vessel using acoustic washing to harvest a spentmedium and place cells in a fresh medium according to one embodiment ofthe present invention.

FIGS. 19A-19C illustrate a diagram of an aptamer selection from alibrary, multiplexed beads or cells with target molecules incubated withaptamer library according to one embodiment of the present invention.

FIG. 20 illustrates an example of a dual stage acoustic valve sorteraccording to one embodiment of the present invention.

FIGS. 21A and 21B illustrate a system and method for optical analysis ofacoustically repositioned particles and a medium.

FIG. 22 illustrates a diagram of particle groupings with differentparameters.

FIG. 23 illustrates an image of acoustically repositioned particlesimaged by an imager.

FIG. 24 illustrates acoustic positioning of particles for fusion orreaction.

FIG. 25 illustrates a first acoustic focuser focusing particles in atight, single file line and then a second acoustic focuser separatingparticles based on size.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to systems using acoustic radiationpressure. Acoustic radiation pressure can be used to concentrate andalign particles in fluids. This ability has many applications in thefields of particle analysis and sample preparation. As described hereinacoustic radiation pressure is applied primarily to flow cytometry,reagents for use in flow cytometry and sample preparation for flowcytometry. Acoustic cytometers, using relatively large dimension flowchannels, concentrate particles from the entire volume of the channel toa small acoustic trap in the center of the channel and can thereforeoffer both controllable flow and high particle analysis rates withoutrequiring highly concentrated samples. Many of the sample preparationmethods have wider application and a few of these embodiments aredisclosed.

As used herein “acoustic contrast” means the relative difference inmaterial properties of two objects with regard to the ability tomanipulate their positions with acoustic radiation pressure. Theacoustic force due to acoustic radiation pressure on a compressible,spherical particle of volume V in an arbitrary acoustic field(neglecting viscosity and thermal conductivity) can be written in termsof an acoustic radiation pressure force potential U:

$\begin{matrix}{U = {\frac{4}{3}\pi\;{{a^{3}\left\lbrack {{\left( {\beta_{o}\frac{\left( p^{2} \right)}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{\rho_{o}\left( v^{2} \right)}{2} \right)f_{2}}} \right\rbrack}.}}} & (1)\end{matrix}$Here, a is the particle radius, β₀ is the compressibility of thesurrounding fluid, and ρ₀ is the density of the surrounding fluid. Thepressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f₁ and f₂ are thecontrast terms that determine how the mechanical properties(compressibility and density) of the particle differ from the backgroundmedium. They are given by:

$\begin{matrix}{f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}} & \left( {2a} \right) \\{f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{2\left( {\rho_{p} + \rho_{o}} \right)}} & \left( {2b} \right)\end{matrix}$The subscript p corresponds to intrinsic properties of the particle. Theforce F acting on a particle is related to the gradient of the forcepotential U by:F=−VU  (3)Particles will be localized at positions where the potential U displaysa minimum (stable equilibrium). The acoustic contrast of a particle (ormedium or fluid) is determined by the density and compressibilitydifferences between it and the background medium or fluid as defined byterms f₁ and f₂ in Eqs. 2a and 2b. The relative magnitudes and signs off₁ and f₂ determine the behavior of the radiation force potential U andthus determine the magnitude and direction of the acoustic radiationpressure force. As an example, if a particle and the background mediumor fluid share the same density value (ρ_(p)=ρ₀), then f₂ is zero andthe acoustic contrast is due only to compressibility differences in f₁.If both f₁ and f₂ are zero, then acoustic contrast is zero. Viscosityand thermal conductivity will also have effects on acoustic contrast,but theses are widely neglected in the literature. Equation 1 isgenerally sufficient to describe the acoustic contrast relationship formost samples of interest.

As used herein “a” means one or more.

As used herein “assaying” means a method for interrogating one or moreparticles or one or more fluids.

As used herein “assay” means a product, including but not limited to, anassay kit, data and/or report.

As used herein “flow cell” means a channel, chamber or capillary havingan interior shape selected from rectangular, square, elliptical, oblatecircular, round, octagonal, heptagonal, hexagonal, pentagonal, andtriagonal.

As used herein “channel” means a course, pathway, or conduit with atleast an inlet and preferably an outlet that can contain an amount offluid having an interior shape selected from rectangular, square,elliptical, oblate circular, round, octagonal, heptagonal, hexagonal,pentagonal, and triagonal.

As used herein “acoustically focusing”, “acoustically focused”,“acoustically focuses” and “acoustic focusing” means the act ofpositioning particles within a flow cell by means of an acoustic field.An example of acoustic focusing of particles is the alignment ofparticles along an axis of a channel. The spatial extent of the focalregion where particles are localized is determined by the flow cellgeometry, acoustic field, and acoustic contrast. As viewed in the crosssectional plane of a flow cell, the shape of observed focal region canresemble a regular geometric shape (e.g. point, line, arc, ellipse,etc.) or be arbitrary. The primary force used to position the objects isacoustic radiation pressure. The acoustic systems of the presentinvention are sometimes referred to herein as flow cytometers, acousticcytometers, flow cells or long transit time devices, however all suchsystems have acoustic radiation pressure.

As used herein “acoustically reorienting” and “acoustically reorients”means the act of repositioning the location of miscible, partiallymiscible, or immiscible laminar flow streams of fluid or medium within adevice with acoustic radiation pressure. This technique utilizesdifferences in the mechanical properties (acoustic contrast) of separatelaminar streams in a flow channel. When two fluids are brought intocontact, a large concentration gradient can exist due to differences intheir molecular makeup's resulting in an interfacial density and/orcompressibility gradient (acoustic contrast between streams). Fordiffusion, time scales that are larger than the time scales of theacoustic excitation, the laminar flow streams can be acted upon withacoustic radiation pressure. Under the action of the acoustic field, thestreams are reoriented within the flow cell with an acoustic field basedupon their acoustic contrast.

As used herein “particle” means a small unit of matter, to include butnot limited to: biological cells, such as, eukaryotic and prokaryoticcells, archaea, bacteria, mold, plant cells, yeast, protozoa, ameba,protists, animal cells; cell organelles; organic/inorganic elements ormolecules; microspheres; and droplets of immiscible fluid such as oil inwater.

As used herein “analyte” means a substance or material to be analyzed.

As used herein “probe” means a substance that is labeled or otherwisemarked and used to detect or identify another substance in a fluid orsample.

As used herein “target” means a binding portion of a probe.

As used herein “reagent” is a substance known to react in a specificway.

As used herein “microsphere” or “bead” means a particle having acousticcontrast that can be symmetric as in a sphere, asymmetric as in adumbbell shape or a macromolecule having no symmetry. Examples ofmicrospheres or beads include, but are not limited to, silica, glass andhollow glass, latex, silicone rubbers, polymers such as polystyrene,polymethylmethacrylate, polymethylenemelamine, polyacrylonitrile,polymethylacrylonitrile, poly(vinilidene chloride-co-acrylonitrile), andpolylactide.

As used herein “label” means an identifiable substance, such as a dye ora radioactive isotope that is introduced in a system, such as abiological system, and can be followed through the course of a flow cellor channel, providing information on the particles or targets in theflow cell or channel.

As used herein “signaling molecule” means an identifiable substance,such as a dye or a radioactive isotope that is introduced in a system,such as a biological system, and can be used as a signal for particles.

As used herein “inherently axially symmetric” means an object thatdisplays a high degree of axial symmetry. Examples of inherently axiallysymmetric geometries include oblate circular cross section cylinders,elliptical cross section cylinders, and oval cross section cylinders,but not limited thereto.

Field based focusing of particles via magnetic fields, optical fields,electric fields and acoustic fields, enables the localization ofparticles without the need for sheath fluid. Focused particles can beflowed past interrogating light sources at whatever linear velocity ischosen using an adjustable external pumping system such as a syringepump. Field based focusing also concentrates particles in the mediumwhich allows for high particle analysis rates without the need topre-concentrate samples.

Field based focusing for flow cytometry, where particles are analyzedone by one, has been accomplished with dielectrophoretic and acousticsystems. This can be done using other fields, such as magnetic fields,optical fields or electrophoretic fields.

Field based focusing of particles relies on contrasts in physicalproperties between the particle being focused and the medium. Fordielectrophoretic focusing, this relies on dielectric properties. Formagnetic focusing, magnetic susceptibility and for acoustic manipulationthis relies on acoustic properties, primarily density andcompressibility.

Magnetic focusing of cells typically requires binding of magneticmaterial to the cells and dielectrophoretic focusing typically requirescareful control of the media conductivity as well as very smalldimensions for high field gradients. This makes acoustic focusingparticularly attractive for many analytes as it typically does notrequire reagents to change the contrast of particles and can beperformed in relatively large dimension channels with complex one ormore media of highly variable conductivity and or pH.

Referring now to FIG. 1, a schematic comparison of planar microchannelfocusing 103 and 105 and line driven capillary focusing 107 and 109.Planar focusing results in a two dimensional sheet of particles 102 withvarying velocities arrows along the flow direction. Cylindrical linedrive focusing places particles 102 in the center where they travel atthe same rate single arrow.

Particle Manipulation in Acoustically Driven Capillaries

To calculate the acoustic force on particles within an ultrasonicstanding wave, the acoustic radiation pressure force on a compressible,spherical particle of volume V in an arbitrary acoustic field can bewritten in terms of an acoustic radiation pressure force potential U(Gor'kov 1962):

$\begin{matrix}{U = {\frac{4}{3}\pi\;{a^{3}\left\lbrack {{\left( {\beta_{o}\frac{\left( p^{2} \right)}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{\rho_{o}\left( v^{2} \right)}{2} \right)f_{2}}} \right\rbrack}}} & (1)\end{matrix}$

Here, a is the particle radius, β₀ is the compressibility of thesurrounding fluid, and ρ₀ is the density of the surrounding fluid. Thepressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f₁ and f₂ are thecontrast terms that determine how the mechanical properties of theparticle differ from the background medium. They are given by:

$\begin{matrix}{f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}} & \left( {2a} \right) \\{f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{2\left( {\rho_{p} + \rho_{o}} \right)}} & \left( {2b} \right)\end{matrix}$

The subscript p corresponds to intrinsic properties of the particle. Theforce F acting on a particle is related to the gradient of the forcepotential U by:F=−VU  (3)

Particles will be localized at positions where the potential U displaysa minimum.

According to one embodiment of the present invention a round or oblatecross-section capillary that acoustically focuses particles either alongthe axis of the capillary or along the capillary wall is tuned with anacoustic wave. The position of the particle within the capillary dependsupon the value of its density and compressibility relative to thebackground medium as shown in the acoustic contrast terms f₁ and f₂above.

A cylindrical geometry according to one embodiment of the presentinvention creates a radial force profile with radial restoring forcesthat hold the particles in a single stream line along the axis of theflow. This affords single file particle alignment along the axis of thecapillary while using only a single acoustic excitation source. Thereare several benefits identified with the cylindrical (inherently axiallysymmetric geometries such as oblate cylinders, ellipses, etc.) forparticle manipulation. The benefits include: higher throughput on theorder of several ml/min versus several hundred μl/min per fluid channel.Fine positioning of blood cells along the axis of a capillary at flowrates of 0 to 5 mL/minute have been achieved in 340 micron diametercapillaries.

Referring now to FIG. 2, a single line acoustic focusing apparatus isillustrated according to one embodiment of the present invention. FIG. 2gives a side view A and axial view B of a cylindrical tube 201 acousticfocusing device that acoustically focuses particles 203 to a pressureminimum in the center of the tube 209 by transducer 205. The stream ofparticles is sent to analysis 211. Analysis includes any post focusinginterrogation or further processing. The flow cell is not limited to atube or a cylindrical shape.

The particles are maintained in a single velocity stream line thatallows uniform residence time for similar size and acoustic contrastparticles. This is important for any process for which reaction kineticsare important.

Radial force driven acoustic focusing of particles coupled with tightcentral focusing of a light source on the particles allows analysis ofparticles one by one as in a flow cytometer and simultaneousconcentration of the particles. This type of analysis is much morepowerful than a simple fluorescent readout step as it allows multiplexedidentification and quantification of each particle/assay as well assingle particle statistics.

Acoustic Manipulation of Background Media

According to another embodiment, a method for acoustically reorienting amedium provides that the medium within the device is acousticallymanipulated in addition to the position of the particles. Thisembodiment utilizes differences in the mechanical properties (acousticcontrast) of separate laminar streams in a flow channel. As used herein,medium is used interchangeable with fluid.

Referring now to FIG. 3, a schematic of a line driven capillary 301acoustically focusing particles to the center of a flowing fluid stream311 comprising clean fluid 307 as the particles move across laminar flowlines 315 is illustrated according to one embodiment of the presentinvention. Particles in sample 305 are acoustically focused from samplestream 309 and can be tightly acoustically focused for single fileanalysis. Wash buffer 307 provides fluid stream in which particles arefinally contained. Transducer 330 provides acoustic standing wave.

Referring now to FIG. 4, one embodiment of an acoustically drivencapillary 401 and 405 with two laminar flow streams 403 and 407 incontact is illustrated in FIG. 4A. Upon activation of the acoustic fieldin FIG. 4B, the positions of the fluid streams are acousticallyreoriented based upon the acoustic contrast of each stream. In oneembodiment, the flow stream with greater acoustic contrast 403 b isreoriented to the center of the acoustically driven focused capillarywhile the flow stream with lower acoustic contrast 407 b is acousticallyreoriented near the capillary walls. In FIG. 4A, the acoustic field isOFF and streams flow parallel down the channel. As illustrated in FIG.4B, when the acoustic device is activated in a dipole mode, Stream 1 403a moves coincident with the central axis of the capillary partiallydisplacing Stream 2 407 b. Equations 1-3 approximate the stream that ismore dense and/or less compressible is forced to the central axisposition. Flow direction is downward on the plane of the page.

Equations (Eqs.) 1 and 2 describe an acoustic contrast that predicts themagnitude and direction of the acoustic radiation pressure force onparticles in a fluid or medium. The force depends upon the differencesin the density and/or compressibility of a particle relative to thedensity and/or compressibility of the background medium. Although thistype of effect has traditionally been used to study particles,emulsions, and bubbles in fluids, it has also been applied to extendedobjects in fluids. For example, the acoustic radiation pressure forcehas been shown to effectively stabilize liquid bridges of silicone oilin water. It was observed that liquid bridges density-matched to abackground water medium can be driven with modulated acoustic radiationpressure. The force results from a difference in the compressibilities(acoustic contrast) of the liquid bridge and background medium.Similarly, experiments using air as a background medium have proven theacoustic radiation force is effective for the manipulation of both smalldiffusion flames of natural gas and dense gases surrounded by air.

The effect shown in FIG. 4 takes advantage of differences in thecomposition of the laminar flow streams. The streams can be immiscible,partially-miscible, or miscible. When two fluids are brought intocontact, a large concentration gradient can exist due to differences intheir molecular makeups. For immiscible fluids, this interface isassumed to be infinitely narrow. For miscible fluids, the concentrationgradient is a transient interfacial phenomena that relaxes over time dueto diffusion and other transport mechanisms. For the description ofacoustic processes, the concentration gradient is viewed as a densityand/or compressibility gradient. For diffusion time scales that are muchlarger than the time scales of an acoustic excitation, the laminar flowstreams can be considered isolated entities with different densities andcompressibilities (acoustic contrast) that can be acted upon withacoustic radiation pressure. Multiple laminar stream systems have beendeveloped where the flow streams are manipulated consistent with thedensity and compressibility relationships shown in Eqs. 1 and 2.Examples of these systems are illustrated herein.

It should be noted that Eq. 1 is approximated in the long wavelengthlimit, where it is assumed that the particle acted upon by the acousticradiation pressure force is much smaller than the wavelength of soundexcitation (λ>>a). It also ignores multiple scattering from theparticle. (Contributions from wave reflections at the media interfacesto the resident acoustic field can also become considerable as theacoustic contrast between streams increases.) For this reason, it isassumed that Eq. 1 is not an exact description of the interaction of theacoustic field with the laminar flow streams in the devices describedhere. Acoustic radiation pressure induced manipulation of misciblelaminar flow streams of diameter b in the limit where λ>>b is upheld, aswell as for larger diameter streams where λ˜4 b, have been observed.Equations 1-2 serve as qualitative predictors for the location of thefinal stream position by defining a relationship between the relativedensity and compressibility of the streams within the flow channel.Corrections to the final shape of the streams due to shaping associatedwith acoustic radiation pressure and gravity will affect their finalcross sectional geometry within the cavity, but the approximate positionof the stream is still predicted by density and compressibilitycontrasts (acoustic contrast).

FIGS. 5A-5D illustrate the separation of micron sized polystyrenefluorescent orange/red particles from a background of nanometer sizedgreen particles in homogeneous media according to one embodiment of thepresent invention. The time-averaged acoustic force scales with thevolume of a cell/particle. Because of this it is possible to fractionateparticles not only by acoustic contrast to the media but also by size.By flowing a clean stream in the radial center of a separation devicehowever, it is possible to prevent the smaller particles from reachingthe center before the point of axial particle collection. Furthermore,if the center stream has higher specific gravity and/or lowercompressibility than the outer sample stream, the particles/cells withgreater acoustic contrast than the center wash fluid will continue tofocus to the capillary axis while particles/cells of lesser contrastwill be excluded.

Referring now to FIG. 5A, polysciences fluoresbrite polychromatic red5.7 μm latex particles are mixed with Polysciences 200 nm fluoresbritegreen particles in the coaxial stream. A particle stream flowing throughthe capillary under epi-fluorescent illumination (FITC long-pass filter)with acoustic field off is illustrated. FIG. 5B is an activation of theacoustic field that acoustically focuses the 5.7 μm particles (whichfluoresce yellow under blue illumination) to a line along the centralaxis of the capillary, leaving the 200 nm particles not acousticallyfocused and remain in their original flow stream. The 5.7 μm particlesare like particles with like acoustic contrast. FIG. 5C shows greenillumination with red band pass filter. The 5.7 μm particles fluorescered while the 200 nm particles are not excited. FIG. 5D illustratesclean core stream 507 introduced alongside coaxial stream containingfluorescent background 505. Transducer 503 includes acoustic standingwave (not shown). Particles 509 are acoustically focused upon enteringstanding wave. The acoustically focused particles cross from samplestream 509 to core stream 507 and thereby are removed from sample fluid.

Referring now to FIG. 6, particle acoustic focusing of particles acrosslaminar streams and acoustic reorientation of medium is illustrated.FIG. 6A illustrates the fluorescence image of an optical cell coupled tothe end of an acoustic focusing cell with acoustic field off. Whitelines are added to indicate edges of 250 μm flow cell. Excitation lightpasses through a 460 nm bandpass filter and emission is filtered througha 510 nm long-pass filter. Flowing through the bottom half of the flowcell is a mixture of 10% whole blood in PBS buffer spiked with 25 μg/mlof R-Phycoerythrin fluorescent protein (orange fluorescence). Whiteblood cell DNA is stained with SYTOX green. At the top is 6% iodixanolin PBS buffer (dark).

FIG. 6B illustrates the same optical cell and media after acoustic fieldis turned on. The 6% iodixanol in PBS buffer acoustically reorients tocenter while the PBS/PE/blood plasma mixture is acoustically reorientedtoward both the left and right sides of the cell (top and bottom in thefigure). The white cells leave their original medium and areacoustically focused to the center where they are observed as a greenline. Red cells also acoustically focus to this location but are notvisible in the fluorescent image.

FIG. 6C illustrates MATLAB plot of the approximate acoustic forcepotential (Eqs. 1 and 2) for particles that are more dense/lesscompressible than the background. This is an axial view of anacoustically driven capillary with an extended source aperture (flow isinto the page). More dense, less compressible particles/media e.g. cellsand iodixanol/PBS medium, are acoustically focused/acousticallyreoriented toward the center (dark blue region) and less dense and/ormore compressible media e.g. PBS/PE/blood plasma mixture areacoustically focused/acoustically reoriented toward the left and rightsides (dark red regions).

When separating particles or cells using heterogeneous media in whichthe wash stream fluid's specific gravity and/or compressibility(acoustic contrast) differs from that of the sample fluid stream, theseparate laminar streams can be affected by the acoustic field. Forexample, if blood cells are to be separated from the protein in serumand the wash stream has higher specific gravity/lower compressibility,then the entire sample stream is pushed toward the center of the fluidcavity (e.g. capillary axis in an acoustically driven capillary). Thiscondition is met when even very dilute blood in physiological saline isthe sample stream and physiological saline is the wash stream. Ifhowever, the wash stream is made to have higher specific gravity/lowercompressibility than the sample stream, but lower specificgravity/higher compressibility than the cells, the wash remains in thecentral core, the cells move toward the center of the cavity and thesample medium is pushed to the sides.

Modeling for the acoustic field distribution shows where the samplemedia should approximately be positioned for the case described above(FIG. 6C). Using Eq. 1-3, a potential minimum exists in the center ofthe capillary for particles (or approximately for flow streams) thatpossess higher specific gravity/lower compressibility. Conversely,particles (or flow streams) that possess lower specific gravity/highercompressibility will be positioned at the potential maxima in the figureas the sign is reversed in Eqs. 2. An interesting result occurs when asample stream of lower density (and/or higher compressibility) is flowedalong the axial center of the capillary and a higher density (and/orlower compressibility) fluid stream is flowed adjacent to it. Thestreams will acoustically reorient to comply with the potential shown inFIG. 6C. This kind of stream separation has not been demonstrated orreported in planar systems.

The ability to place samples in the central core stream and stillseparate the sample fluid from the cells or particles can be used toincrease throughput. The acoustic force is strongest near the minimumpotential U in FIG. 6C and the distance the particle must travel to theminimum is minimized.

The data in FIG. 6 shows that phycoerythrin in the acousticallyreoriented streams is positioned further from the center of the flowstream than with the acoustic field turned off. This may be used toadvantage in an in-line system designed to exclude free antibody orother species, for example, particularly for slow flow rates/longresidence times where diffusion might otherwise significantly penetratethe wash stream.

Referring now to FIG. 7, analysis in a flow cytometer like configurationwhere cells/particles are paraded through a tightly focused laser,illustrated such that the laser can be focused so that it does notexcite the “dirty” media (FIG. 7A). Alternatively, the clean stream canbe flowed independently through the optics cell (FIG. 7B). For thismethod clean media and target cells reach the detection region but theparticles may need refocusing by a second focusing element. If thesample is injected slightly to one side of center and the stream is asmall enough fraction of the total flow, the sample stream can beconfined to one side of the optics cell (FIG. 7C). This is advantageousin flow cytometry for separating free vs. bound flourophores in theanalysis region.

Referring now to FIG. 7A, an embodiment of the present inventioncomprises sample 715 a which is introduced into the system alongsidewash buffer 713 a. Particles 712 in sample 715, sample 715 and washbuffer 713 are introduced into capillary 703 a. A line drive 701 oncapillary 703 introduces acoustic standing wave (not shown) naming auser defined mode (dipole mode is this example). The sample 715 a andbuffer 713 a are acoustically reoriented and particles are acousticallyfocused based upon the acoustic contrast of each. Acoustically focusedparticles 717 are transited to an interrogation point 716 where laser717 impinges electromagnetic radiation. An optical signal from theinterrogated sample 719 is detected by the detector 705. The detectormay be a PMT array for example.

Referring now to FIG. 7B, sample 715 comprising particle 702 isintroduced into the system. The sample 715 a, wash buffer 713 a andparticle are introduced into capillary 703. An acoustic wave (dipolemode) is induced into the capillary 703 by a first acoustic waveinducing means 701 such as a PZT drive but not limited thereto as otheracoustic wave inducing means will produce same standing wave. Acousticfocusing of particles 702 cause each particle to be acoustically focusedsuch that each particle having high enough acoustic contrast will focusin a line 717. Sample buffer with a lower concentration of particlesafter acoustic focusing will be discarded to waste 721 buffer 713 and717 particle will be transited to a second acoustic wave inducing means714. Particles are interrogated with a laser 709. The optical signal 719for interrogated sample is sent to detector 705.

Referring now to FIG. 7C, sample 715 comprising particle 702 and buffer713 are introduced into the system. Sample 715 flow next to capillarywall 703 and buffer 713 a flows against the opposite wall. Transducer717 induces acoustic wave that acoustically reorients sample 715 b,acoustically focuses particles 714 and acoustically reorients buffer 713b. The particles 714 are transited to the interrogation point forinterrogation of the particles 714 and buffer 713 b by laser 709. Theoptical signal from interrogated particle 714 and/or buffer 713 b isdetected by detector 705. The velocity of the sample stream, bufferstream, particles is controlled by pumping system (not shown) such thatthe velocity is variable between 0 meters/second to 10 meters/second inthe forward, reverse or stopped direction. Particles are washed in anacoustically reoriented first fluid which replaces the second fluid toproduce washed particles.

One aspect of one embodiment of the present invention provides for anacoustic particle focusing technology in a cytometer that is capable ofboth high particle analysis rates up to 70,000 particles/second and/orcapturing images from user selected subpopulations of cells.

Another aspect of one embodiment of the present invention provides for asystem and method to analyze more than one hundred thousand cells perminute using traditional flow cytometry measurements and periodicallyadjust the velocity of the focused stream to collect images of onlythose cells that meet user defined criteria.

A further aspect of one embodiment of the present invention provides fora system and method wherein a first and a second fluid are acousticallyreoriented and wherein the second fluid suppresses non-specific bindingof a reagent that binds to a population of the particles.

Yet another aspect of one embodiment of the present invention providesfor a system and method wherein particles are acoustically reorientedfrom a first fluid to a second fluid. The second fluid has a higherconcentration of particles suspended therein after acoustically focusingthe particles as compared to the second fluid prior to acousticallyfocusing the particles. Acoustically focusing the particles preferablycreates a line of particles through about a center axis of a channelthat flows parallel to an axis of flow.

FIG. 8 illustrates an embodiment of the present invention comprising aschematic of an acoustically focused flow cell for acousticallyorienting particles and flow streams prior to collecting theacoustically focused sample. The sample 801 is introduced into a flowcytometer 850 that contains a transducer 831 for acoustically focusingparticles 832 prior to analysis. Sample container 801 comprising sampleparticles 803, 807 and 809 is introduced to a flow cell 810. Atransducer 811 provides acoustic wave to flow cell 810 to producedacoustically focused particle 815. Wash or other reagent 802 inintroduced to flow cell 810. Particles 809 and 807 are acousticallyfocused into stream 805. The acoustically focused particle 815 existswith the wash stream 815 and is collected at collection/incubation site819. Wash stream 821 is introduced from wash 817. Flow cell 810 b withacoustic field generator 822 receives particles 823. The particles areacoustically focused 825 prior to introduction into the focus cytometer850. A transducer 831 provides to flow cell 851 acoustic field andparticles are acoustically focused 832 prior to reaching aninterrogation point 852. Interrogation light 833 impinges on particle. Asignal 854 from impinged particles is sent to detector 835 for analysis.The particle flows through system to point 837 for collection. In thisembodiment, sample particles 803, 807 and 809 preferably have a particleacoustic contrast that is different from the acoustic contrast of samplecontainer 801.

Advantages of Controllable Flow

The ability to control the linear velocity of particles in a stream fora field focused system while maintaining high particle analysis rates inrelatively large dimension channels enables improved analysis ofparticles and practical analysis of particles in ways that werepreviously not feasible.

By using lower linear velocities than conventionally used in flowcytometry and allowing each particle to spend longer times in theinterrogation light, for example a laser, one can achieve the extremelyhigh sensitivity seen in slow flow hydrodynamic systems without thedrawbacks of clogging and low throughput. In addition, the utility ofmarkers that are not typically used in cytometry because of fast transittimes can be greatly increased. Among these markers are luminescenceprobes such as lanthanides and absorptive dyes such as cytologicalstains and trypan blue. Imaging of particles is also much more easilyachieved by using slow flow or even stopped flow without resorting tospecialized tracking technology like that used in imaginghydrodynamically focused particles (Amnis, Seattle, Wash.).

While nearly all labels currently used in cytometry would benefit fromlower laser power to reduce photobleaching and non-radiative states andthe longer integration of signal afforded by longer transit times, somethat will benefit more than others are discussed herein. These includefluorophores/luminophores that have long lifetimes and or low quantumyields/extinction coefficients. Most chemi bioluminescent species alsobenefit tremendously from longer transit times as their energy is givenoff on time scales much greater than those used in conventionalcytometry analysis. Long lifetime labels are for example labels withlife times greater than about 10 ns. For example: labels with life timebetween about 10 ηs to about 1 μs, labels with life times between about1 μs to about 10 μs labels with life times between about 10 μs to about100 μs, or labels with life times between about 100 μs to about 1 ms andabove.

One aspect of the present invention provides for controllable linearvelocity ranging from 0 m/s to 10 m/s without compromising core diameterand particle concentration. In a preferred embodiment the linearvelocity is in the range of about 0 m/s to about 0.3 m/s. In a morepreferred embodiment the linear velocity is in the range of about 0.3m/s to about 3 m/s. In a more preferred embodiment the range is betweenabout 3 m/s to about 10 m/s. Referring now to FIG. 9, a flow diagramillustrating one embodiment of the present invention is illustrated. Afield based means 905 focuses particles into a line or plane, preferablyacoustically. The particles are transited through the system preferablyby a pumping system 903 that can be adjusted to the desired flow ratefor the desired linear velocities. A means for optical excitation 907 ofthe particles and a means for collection and analysis 909 offluorescent/luminescent light given off by the sample comprising theparticles. Average linear fluid velocity is given by the flow ratedivided by the cross sectional area but particles will generally travelat nearly the same speed as the fluid lamina they are in. Particlesfocused to the center of a channel for most channel geometries usedwould travel about twice the average velocity. Preferably, the systemprovides possible pulsed or modulated excitation at slower rates, datasystems to accommodate longer transit times and slower pulse rates andreduced waste that can readily be run again or transferred to anotherprocess 911 without concentration.

Slowing Linear Velocities—Single-Line Focused System

One embodiment of the present invention comprises a method to improvesignal is by increasing the number of photons given off by afluorescent/luminescent label by illuminating it for a longer timeperiod with a continuous light source. and particles with a linearvelocity of 0.3 m/s, this number increases over the prior art by about10 fold. At this velocity and assuming an average of 100 micronsdistance between particle centers, about 3000 particles per second canbe analyzed. It is the combined ability to focus particles andconcentrate them that allows these long transit times for high particleanalysis rates. If the velocity is further decreased to 0.03 m/s, 100fold more photons would be given off and 300 particles per second couldbe analyzed.

In another example, semiconductor nanocrystals also referred to asquantum dots are highly resistant to photobleaching, so the gainspredicted in the above example might not be so dramatic for otherfluorophores that are prone to photobleach. All fluorophores however,are limited in continuous excitation by a power threshold that achieves“photon saturation” by exciting a maximum number of the fluorophores atany given time. Any more excitation photons will not produce any morefluorescence and will in fact decrease fluorescence by increasingphotobleaching or exciting to non-radiative states. Often, one mustbalance excitation power with photobleaching rate and non-radiativestate excitation such that the most fluorescence is emitted for a giventransit time. In short, longer transit times will yield more photons fora given excitation power, but reducing light source power can furtheryield more photons per fluorophore during the analysis time. This isparticularly important for high sensitivity applications in which veryfew labels may be bound to the target. Lower laser power also reducesfluorescent background, which can further increase sensitivity andresolution of particle populations.

Slower linear velocities can increase the signal from any label, but italso makes it practical to use dimmer labels and long lifetime labelsthat are not commonly used for lack of photon yield in short transittimes. Lanthanide chelates, for instance, have very long Stokes shiftsand very narrow emission wavelengths so they can be highly specificlabels but they will emit relatively few photons over a short transitperiod. Nanoparticles using europium, for instance, may have lifetimesof about 0.5 milliseconds. In a field focused system, transit times canbe slowed to milliseconds or more allowing several cycles of excitationand emission to be monitored. Downstream optics are not required.

Pulsed or Modulated Excitation

According to one embodiment of the present invention an excitationsource is pulsed or modulated. Many commercial light sources areavailable to do this by affecting the light source itself and usingdigital or analog control. Methods such as chopper wheels andacoustoptic modules can also modulate the interrogation light sourceexternally. For some applications it is also desirable to sync thedetectors with the light source in time such that events can becorrelated to excitation peaks or valleys. Correlating a Rayleighscatter detector that detects light scattered from passing particleswith the fluorescence detector(s) in time is one preferred method.Lock-in amplification can be used to help eliminate electronic noise atfrequencies other than the modulation frequency but averaging toeliminate noise can be accomplished digitally if the data is collectedin a digital format.

Using pulses with relatively long rest times that allow relaxation fromtriplet states can increase the overall fluorescence yield offluorophores vs. equivalent power strong continuous wave excitation.This is again of particular importance to high sensitivity applicationswhere only a few fluorophores are present. For a slow transit system, asin the present invention, particularly a field focused system, 67 pulseswith microsecond timing can be monitored for a 0.3 m/s linear velocityfor probes such as perCP where the triplet state is estimated to beabout 7 microseconds. Pulsed or modulated light sources have theadditional advantage of allowing phased locked amplification oraveraging of time correlated data, either of which will reduceelectronic noise.

Photobleaching of Undesired Fluorescence

One can take advantage of labels that have a high resistance tophotobleaching by strongly illuminating a cell or particle havingundesired fluorescence (often cellular autofluorescence) or the mediumthe particle is in, e.g. serum. The use of long transit times toaccomplish this allows more photobleaching for a given excitation power.While in-flow upstream photobleaching is effective in a long transittime system, it can also be done in the present invention with a singlelight source in a long transit time system by examining the signal asthe cell passes. The fluorescence of the less resistant species willdecrease more quickly than that of the resistant specific label. Thisdecrease not only increases specific signal to noise but the rate ofdecay can also be used to separate the non-specific signal from thespecific signal by allowing a quantitative subtraction of theautofluorescence present. Quantum dots are an example of a good labelfor this purpose not only because of their high photobleachingresistance but because of their long Stokes shift. The Stokes shift canmove the signal out of the primary cellular auto-fluorescence peak whichimproves signal to noise already but it also opens that spectralwavelength for use of an additional detector to monitor theauto-fluorescence. This by itself has been used to subtract cellularauto-fluorescence but the technique could be made more effective by alsomonitoring the decay rate. Decay rate can also be used to compensatebleed-through for different channels (colors) of fluorescence. If, forexample a particle is labeled with both fluorescein and phycoerythrinand is excited with 488 nm light, the relative decay in the green andred channels can be used as a quantitative measure of how muchfluorescein fluorescence is picked up by the red channel. This methodimproves compensation accuracy and eliminates the need for runningcompensation controls.

New Useful Probes

Lanthanide chelates, especially those using europium and terbium havedominated the time resolved probe market. These complexes are generallyexcited by wavelengths shorter than 400 nm but developments in theseprobes, e.g. Eu(tta)3DEADIT (Borisov and Klimant), have resulted incomplexes that can be very efficiently excited by 405 nm light. This issignificant because the low cost, high quality 405 nm diode lasersdeveloped in the entertainment industry promise to lower the cost ofviolet excitation. Many other metal ligand complexes excited at avariety of wavelengths have high potential for use in longer transittime cytometers. The list can be further increased by includingluminescence resonance energy transfer (LRET) probes which generalcombine a long lifetime fluorophore like a metal ligand complex with ashorter lived dye. Lackowiz, Piszczek and Kang (2001) found that in suchcomplexes it was possible to achieve a high quantum yield fluorophore bycombining a low quantum yield metal ligand complex donor with a highquantum yield acceptor by combining a ruthenium ligand complex withshort lifetime dyes.

These types of tandem probes are particularly useful in a long transittime cytometer because the long lifetime of the donor and the shortlifetime of the acceptor combine to give a medium lifetime probe thatwould have too long a lifetime for a conventional cytometer but a shortenough lifetime to increase throughput in a long transit time cytometer.For example, the ultra long lifetime of a terbium complex in a DELFIA™assaying format has a lifetime of 1045 microseconds as compared with aTerbium fluorescein complex in a LanthaScreen™ assaying which has alifetime of 160 microseconds. Lifetime can also be manipulated withchanges to the metal chelating ligands (Castellano et at. 2000). Withnumerous possible metal ligand complex as donors and no limit to thenumber of acceptors many useful probes can be developed on the basis ofspectral and lifetime properties. The multiplex idea can be carried evenfurther using probes having several differing lifetimes e.g. short,medium and long that can be resolved individually by lifetime. A greatadvantage for the lifetime multiplexing scheme is that the samedetectors can be used for overlapping colors, e.g. a fluorescein/terbiumcomplex can be used in conjunction with plain fluorescein.

Qdots® although shorter lived than luminescent probes, have lifetimesthat are long enough (˜10-100 ns) to be well separated from mostconventional fluorophores and short enough to be used in conventionalcytometers but the practical use of lifetimes on this scale has beenlimited. Developments in high speed detectors, lasers and electronicsmake this more practical.

Other luminescent materials such as phosphors and up-convertingphosphors have not achieved success in bioassaying, largely due to theirlarge size. These materials might be very useful however in multiplexbeadsets for cytometry. Their emissions can be distinguished using timeresolved techniques and the up-converting phosphors can be excited usinglong wavelength lasers that would not excite most fluorophores used inassaying.

Secondary Reagents

Secondary reagents using ligands such as biotin, streptavidin, secondaryantibodies and protein A and G will be of particular utility ininexpensive cytometers and long transit time cytometers. For instrumentstaking advantage of violet diodes, availability of violet excited dyesconjugated to the antibodies or other ligands necessary for assaying maybe in short supply, so violet excited secondary conjugates will be veryuseful, e.g. Pacific Blue® or Orange® conjugated to streptavidin/biotinor protein A/G or anti-species specific or probe specific (fluorescein,PE, APC) secondary antibodies can all be used to increase the utility ofan instrument with fewer lasers than are typically necessary to exciteprobes of choice. If, for example, assaying requires antibodies that areonly available as unconjugated or conjugated to Fluorescein or PE orbiotin needed to be performed in a violet only or a violet and redinstrument, protein A or G or species specific secondary reagents can beused for unconjugated antibodies, labeled streptavidin for the biotinantibodies and anti-fluorescein or PE for the dyed antibodies. Qdots®are also excellent examples of violet excited labels that are typicallyused in a secondary format, usually streptavidin conjugates. Their broadexcitation spectrum makes them particularly suited for a single violetlaser system or a violet/red laser system where inter beam compensationcan be minimized.

With acoustic washing, secondary labeling can be accomplished veryquickly and easily by primary labeling, acoustic washing, secondarylabeling and a second acoustic wash. An automated system or semiautomated system to do this will reduce not only assaying time butoperator error.

Secondary long-lifetime labels are particularly suited to a long transittime cytometer with modulated or pulsed excitation as they allow addingthe lifetime parameter for analysis using commonly availableantibodies/ligands.

Other Long Lifetime Methods

For bio/chemi/electro luminescence one can use a pulsed/modulated systemthat analyzes the level of luminescence in between pulses and subtractedthis from the fluorescence for short-lived labels. This luminescencemight be measured using reagents internal to the cell or can be membranebound enzyme labels that interact with substrate added to the sample.Acoustic washing just prior to analysis could ensure that luminescencefrom the medium could be associated with the proper cell. Monitoringenzyme cleaved substrates in a more conventional manner after sorting isanother possibility for drug discovery assaying but it can also beapplied to low level marker assaying that require enzyme amplificationfor detection.

One embodiment of the present invention comprises a method for measuringchemi, bio or electro luminescence in an acoustic particle analyzer. Inthis method, particles capable of producing a chemi, bio or electroluminescence are moved through a channel and are acoustically focusedusing acoustic radiation pressure. The particles are then passed througha zone for collection of luminescence and collect light from theparticle produced from chemi, bio or electroluminescence. In thisembodiment, the particles are preferably focused with a radial acousticfield. Luminescence is preferably collected between excitation pulsesfrom a light source.

Time-Resolved Fluorescence/Luminescence

Another embodiment of the present invention provides a method forcircumventing background fluorescence using probes that continue to emitlight for some time after the background fluorescence has substantiallydecayed. The advantages of slower linear particle velocity make thetechnique much more attractive. This method uses a modulated or pulsedlaser as above but light is also collected and correlated in time to theexcitation valleys where there is little or no excitation light. Thelonger time intervals that are not implemented in conventional flowcytometry cost much less with lower cost lasers and electronics, buttheir primary advantages lie in the ability of maximizing fluorophoreoutput and to use very long lifetime probes such as lanthanide chelatesand lanthanide energy transfer probes. Pulsing at the very slow (forflow cytometry) rate of a thousand times per second with a 10microsecond pulse, would for a transit time of 10 milliseconds forexample, allow 10 cycles of excitation and luminescence collection inwhich virtually all of the luminescence decay of a europium chelatecould be monitored. This pulse rate with a conventional cytometertransit time would allow >90% of the particles to pass without everbeing hit by the laser. If the pulse rate were increased to 100kilohertz with a 1 microsecond, pulse there would still be nearly 9microseconds in which to monitor the lanthanide luminescence as mostfluorophores have 1-2 nanosecond lifetimes and most autofluorescencedecays within 10 nanoseconds. For some lanthanides specificity canfurther be increased by monitoring fluorescence of different emissionpeaks.

The most commonly used lanthanides, terbium and europium, are primeexamples. For the Seradyn europium particles for example, their twoprimary emission peaks are at 591 and 613 nm. The ratio of these peaks(˜13) is a highly definitive signature of this label. The peaks can bereadily distinguished from each other as their bandwidth is so narrow(90% bandwidth for the 613 nm peak at 25 nm) an additional degree ofspecificity could also be achieved from tracking the kinetics of thisemission as the 591 nm peak has a shorter lifetime than the 613 nm peakand the rate of change of the ratio would be extremely specific. In apreferred embodiment increasing luminescence of the label is monitoredduring subsaturation excitation pulses to monitor specificity. If thelabels are not excited to saturation and if the dead time between thepulse is less than the fluorescence decay, each successive excitationcycle would increasingly excite more labels before the decay of otherexcited labels is complete. This gives an increasing trend in signalthat is specific to the lifetime of the probe. With this method, it isnot specifically the phase or lifetime that is being measured but theincrease in the phase shifted emission. In principle, this can be donewith other labels such as quantum dots but as their decay times are muchshorter (10-100 ns), a much quicker pulse or modulation rate is required(˜10-100 MHz). Careful attention must also be paid to the excitationintensity and formation of triplet states such that the specificityadvantage is not lost by collection of fewer photons.

One of the primary sources of unwanted signals in flow cytometry andlabel based techniques in general is the specific fluorescence ofunbound or non-specifically bound labels. For flow cytometry, squeezingthe sample core size to a very small dimension and optical spatialfiltering alleviate the problem of unbound labels to some degree, butultrasensitive applications often require prewashing of the analytespecies from the unbound labels. This is problematic if the labels arenot of extremely high affinity as they may tend to dissociate from theirtargets once washing disturbs the binding equilibrium or kinetics.

Field based in-line particle washing is used to solve this problem.Laminar flow washing relies on the fact that only particles affectedstrongly enough by the field to move across the laminar boundary willenter the clean fluid. This generally leaves most of the labels behind.According to one embodiment of the present invention, the fluidics areconstructed such that substantially clean fluid reaches the collectionor analysis region. Some clean fluid is discarded with the waste inorder to account for diffusion across the laminar boundary and insurehigh purity.

Using in-line field based washing allows for very small time intervalsbetween the alteration of binding kinetics and the analysis. By placingthe wash step very close to the analysis point, washing can be achievedreadily in fractions of a second. Even for relatively low affinitybinding reactions, background reduced analysis can be done beforesignificant dissociation occurs. This capability is of high importancefor many applications where sensitivity is important, but bindingaffinity is relatively low. Many monoclonal antibodies, synthesizedligands and drug candidates fall into this category. FIG. 10 illustratesthe flow chart in FIG. 9 modified to include in-line laminar washing.FIG. 10 comprises an additional pumping system 1007 used to introducethe wash fluid and fluidics which are modified to extract the cleanedparticles after washing. Laminar wash devices can also be installed inseries to increase purity or to process particles in different media,see example FIG. 8.

As illustrated in FIG. 10, an acoustic focusing device 1019 is modifiedwith wash stream 1007 into which target particles can be focused. Washedparticles can be analyzed within fractions of a second of being washed.For the planar device in FIG. 11, focusing is only in one dimension butthis dimension can be stretched out over a large area to increase flowrates.

FIG. 11 is a planar acoustic flow cell comprising laminar wash fluid1107. Sample containing particles 1109 is introduced into flow cell1101. An acoustic wave is introduced 1105. Particles are acousticallyfocused based on acoustic contrast. Channel tuning is dictated by heightrather than width. This allows high width to high aspect ratio channelswith higher throughput. Different standing waves can be used inaccordance with FIG. 11.

Another embodiment comprises a planar acoustic flow cell wherein theacoustic node is located outside flow cell 1101. In this embodiment,particles 1109 are acoustically focused to the top of the flow cellwhere acoustic wave 1105 is introduced.

Multi-Color Analysis

Most of multiplexing in flow falls into two distinct categories. Thefirst is often referred to as multicolor analysis in which severalmarkers on or in cells are examined simultaneously in order to extractas much information as possible from the cells being analyzed. Probes ofdifferent spectral wavelengths are chosen such that there is maximalexcitation overlap and minimal emission overlap and usually the markersthat are known to bind the fewest labels will be given the brightestprobes in hopes that all markers can be resolved. Tandem probes areuseful such that one or two lasers could be used to excite manyfluorophores with different emission spectra. In practice there isconsiderable spectral overlap between probes and a great deal of effortis expended on subtracting the signal contribution of these overlapssuch that each individual probe is accurately quantified.

One embodiment of the present invention provides a system and method toproduce greater signal to noise resolution of the specific signals andimproved methods for isolating the different probes. First, the use oflonger lifetime probes such as the narrow emission of quantum dots andof lanthanides can be better exploited to extract individual signalswith less compensation. By using pulsed or modulated lasers, thefluorescence lifetime of the probes can be monitored to determine theindividual contributions of different dyes. Even if there isfluorescence contributed to detection channels monitoring shorterlifetime probes, this fluorescence can be subtracted based on thequantity of time resolved fluorescence detected. In an embodiment, theincreased signal generated for the longer transit times by usingnarrower bandwidth filters is utilized. These filters collect less lightbut do a better job of separating fluorescence signals from differentprobes. The band width can be made narrower than in conventional systemsbecause there is more signal to spare. The narrow bandwidth approachcollects the entire spectrum with a prism or grating and multi-elementdetectors. In this case the resolution of the spectrum dictates how muchbandwidth per element is collected. Longer transit times make spectralcollection much more practical.

Multiplexed Assaying

The second form of multiplexing in flow is the use of multiple beadpopulations to encode simultaneous assaying such that each can bedistinguished from each other. Specific chemistry is placed on eachpopulation and then the populations are mixed in a single reactionvessel and are then processed in flow. The distinctive properties ofeach population such as size and or fluorescence color and orfluorescence intensity are then detected to distinguish the beads fromeach other. The assay on each bead must be distinguished from the bead'sintrinsic properties and this is typically done by using a differentcolor fluorescence for the assaying itself. These multiplexed solublearrays may be used in diverse applications in accordance with thepresent invention including but not limited to immunoassaying, geneticassaying, and drug discovery assaying.

One type of soluble bead array uses two fluorescent dyes that are dopedinto the beads in varying concentrations to produce populations withdistinct fluorescent color ratios. Between two colors and tenintensities an array of 100 distinct beads is made. It is sometimesdifficult to resolve all of the beads due to variance in the beads andthe detection systems. Longer transit times and the associated highersignal increase sensitivity and resolution. This means that for anycolor coded multiplex system more intensity levels can be resolved andlower concentrations of dyes or other labels need be used. Quantum dotsare excellent for creation of soluble arrays owing to their narrowspectral emission and their ability to all be excited by a single laser.According to one embodiment of the present invention a system and methodprovides for longer transit times of particles having quantum dots.Beads may be made with much fewer quantum dots. This makes them lessexpensive with a lower background interference for assaying. It alsomakes for more resolvable intensity levels such that greater numbers ofdistinct beads can be made. A set using 4 colors of Q-dots and 5intensities for example yields a set of 625 distinct beads. The same 4colors and 10 intensities yields a set of 10,000.

A difficult problem in multiplexed arrays is distinguishing the codingfluorescence from assaying fluorescence. This is a particular problemwhen high sensitivity is required from a high intensity codedmicrosphere. The methods detailed above for multi-color analysis can allbe used to make this easier. In particular, long fluorescence lifetimeprobes, including but not limited to lanthanide labels with quantum dotarrays and time-resolved fluorescence, may be used. Lanthanide labelsare mostly excited by ultra violet light (europium 360 nm maxabsorption). This allows them to be used in conjunction with quantumdots and a single excitation source (e.g. 375 nm diode laser, pulsed ormodulated for life-time applications). The lanthanides' very narrowspectral emission can also be effectively used for lanthanide basedmicrosphere arrays.

By choosing dyes with different fluorescence lifetimes, arrays can alsobe made based on lifetime alone. If for example both a conventionalshort lifetime UV excited dye and a long lifetime lanthanide dye wereimpregnated at discrete concentrations into bead sets, both dyes can beexcited by a pulsed/modulated UV source and the rate of emission decaywould be specific for each discrete concentration. Neither dye would beexcited by longer wavelength lasers, particularly the blue, green andred lasers most common in cytometry. This allows for monitoring assayingacross a wide range of probe colors. In addition, this lifetime decaymethod can be implemented with a single photodetector and a simplefilter to block scattered light at the UV excitation wavelength. Thelifetime method can also be combined with other multiplexing methodssuch as particle size and or multicolor multiplexing to increase arraysize.

In another embodiment of the present invention, fluorescence isseparated from luminescence by collecting light as the particle travelsin and out of a continuous light source. Fluorescence is collected whilethe particle is illuminated and luminescence just after the particle hasleft the illumination. This can be done with properly spaced collectionoptics or over the entire space using a multi-element detector such asmulti-anode PMT or a charge coupled device (CCD).

In another embodiment of the present invention, coupled with imagingoptics, the CCD above, or another imaging detector can also perform timeresolved imaging on focused particles. This is not done in flowcytometry. In fact any imaging technique that requires longer excitationand or emission exposure than is afforded in conventional hydrodynamicfocusing is possible in a field focused system.

Referring now to FIG. 21A which illustrates an embodiment of the presentinvention, sample 2103 containing particles 2102 is introduced in thesystem. Line drive 2105 induces an acoustic wave and particles 2102 areacoustically focused based upon their acoustic contrast 2115.

Optics cell 2117 receives particles and interrogates each particle at aninterrogation site where interrogation source 2111 impingeselectromagnetic radiation upon particle. An optical signal 2113 fromeach particle and/or sample is collected by 2107.

The signal is analyzed and based upon user determined criteria andoptical signal from a particle, a selected particle or group ofparticles is imaged by an imager 2109. The particle may receive anilluminating light from a light source 2119 for imaging. In FIG. 21A, noimage is acquired and the flow rate 2129 remains unchanged.

The flow rate can be altered once a particle meeting a user definedcriteria is detected at 2107. The particle at 2125 in FIG. 21B is movingslower than particle 2125 in FIG. 21A because the flow rate is decreasedto acquire an image in FIG. 21B. The flow rate is decreased once aparticle having the user defined criteria is detected at 2107. An imageof the particle is acquired by imager 2109 and the image may beilluminated by an illumination source 2119. Once the image is acquiredthe flow rate remains decreased 2107. Alternatively, the flow rate isincreased for improved particle throughput through the system.

Referring now to FIG. 22, a bivariant plot of particles analyzed in asystem as shown FIG. 21 is provided according to one embodiment of thepresent invention. Each particle within a group of particles 2202 aresimilar as to Parameter 1 and Parameter 2. Parameter 1 can be, forexample, forward scatter, side scatter or fluorescence. Parameter 2 canbe, for example, forward scatter, side scatter or fluorescence. The userdefined threshold 2201 indentifies particles that meet a threshold forimaging. A particle having a value for Parameter 1 and for Parameter 2that is greater or lesser than the threshold defined by the usertriggers the imager and is imaged. If the particle meets the userdefined criteria then the flow of the stream carrying the particles isreduced to a rate that allows the imager to capture an in-focus image ofthe particle or particles as the particle transits past the imager 2109of FIG. 21.

Other detection thresholds 2205, 2209 and 2215 can be established forparticles having similar Parameter 1 and/or Parameter 2 values.

Referring now to FIG. 23, is a photograph of blood cells 2303 arecaptured for a stream 2305 that is acoustically reoriented. The streamin the optics cell 2307 is slowed so the imager (not shown) can captureparticles 2303 in focus. To create the image, a line-driven capillary ofinner diameter 410 μm is truncated with an optical cell. The opticalcell is a borosilicate glass cube with an interior circular cylindricalchannel the same diameter as the inner diameter of the line-drivencapillary. The frequency of excitation is approximately 2.1 MHz and thepower consumption of the acoustic device is 125 milliwatts. The cellsare lined up single file coincident with the axis of the capillary. Inthis image, flow is nearly static for imaging purposes, but our recentengineering advances in constructing line-driven capillary prototypeshave proven fine focusing of 5 μm latex particles and blood cells atvolumetric flow rates exceeding 5 mL/minute.

In a preferred embodiment of the present invention, a line-drivencapillary is attached to a square cross-section quartz optics cell. Theinner cavity of the optical cell is circular in cross section and hasthe same inner diameter as the line-driven capillary to extend theresonance condition of the fluid column thereby extending the acousticfocusing force into the optical cell. In operation, the particles arealigned along the axis of the capillary by the acoustic force and fluidflow transports them through the system. The particles first enter theanalysis stage where an incident laser beam excites the particle andattached fluorescent markers. When a target of interest is identified byits scatter and fluorescence signature (user defined), the controlsystem decelerates the flow velocity to a value appropriate for therequired imaging resolution. A flash LED (wideband or UV) thenilluminates the particle to capture the image. Once the image iscaptured, the system re-accelerates the flow in the original directionand analysis continues until another particle of interest isencountered.

To achieve the high analysis rates expected from a traditional flowcytometer analyzer, the embodiment will not capture an image of everyparticle analyzed in the system. Rather, the user will construct asampling matrix of particles from gated subpopulations to define a setof particle images to be captured based upon their scatter andfluorescence signatures. With this hybrid approach, high particleanalysis rates are achievable (in excess of 2000/s to search throughlarge populations of cells while capturing only a representative set ofhigh content images that are correlated with traditional flow cytometryparameters. Images will capture cellular morphology, orientation, andinternal structure (e.g. position and number of nuclei) that will beavailable to the researcher to correlate with localized datadistributions generated by the analyzer. The ability to control flowrate while maintaining particle focus along the axis of the flow streamin the acoustic system is the key component necessary for the selectiveparticle imaging after sample analysis.

Fast coaxial flow streams are not required as with conventionalhydrodynamically sheath-focused systems. This alleviates the need fordifferential pressure or flow based delivery systems reducing totalsystem cost. One of the unique capabilities of acoustically driven flowcells is the ability to select the sample delivery rate. By notaccelerating the particles with the coaxial sheath flow, particletransit times through the laser interrogation region of a flow cytometerare ˜20-100 times longer than conventional hydrodynamically focusedsystems. In comparison, higher sensitivity optical measurements can bemade while retaining similar particle analysis rates. This enables theuse of inexpensive optical components to lower system costs.Additionally, acoustically reoriented sample streams can be operated atslow fluid velocities or even stopped and reversed without degradingalignment of the particle stream within the flow chamber allowing raretargets to be repeatedly analyzed or even imaged.

Imaging is performed commercially in flow cytometry using imaging opticby fluid imaging. The fluid imaging uses deep focus optics withouthydrodynamic focusing to take pictures of cells or particles, or evenorganisms as they pass through a rectangular imaging cell. The methodhas adjustable flow but it is limited to taking pictures of particles infocus so many are missed and magnification power and resolution islimited. One system uses hydrodynamic focusing coupled with electronicCCD panning technology that can track the flowing cells. The system isdesigned to keep cells or particles from being blurred. Imaging ratesfor this system are relatively slow (up to 300 cells/sec) but slowerflow and the tracking technology allow long integration times that keepsensitivity high and allow good spatial resolution (up to 0.5 microns).Unfortunately, this technology is very expensive and is limited by thehydrodynamic focus.

Acoustic cytometry of the present invention, in which hydrodynamicfocusing of target cells or particles is replaced or partly replaced byacoustic radiation pressure, adjusts the linear velocity of cells orparticles transiting an interrogation laser while maintaining tightparticle focus. Therefore, light from photoactive probes can becollected for much longer times than are normally possible inhydrodynamically focused cytometers without loss of precision from poorparticle focus. Flow in an acoustic cytometer can even be stopped orreversed, allowing very long observation or imaging for resolution ofspatial information.

One advantage that field focused systems have over hydrodynamic focusedsystems, is again the control of linear velocity while maintainingparticle focus and high analysis rates. Greater transit times can beused all the way up to stopped flow. Pulsed flow is a viable option infield focused systems in which fluid delivery is triggered by upstreamdetectors such that cells or particles are stopped in the imaging regionand flow is maximized when no particles are present. This methodincreases throughput while maximizing exposure times.

Another embodiment of the present invention provides a method toincrease throughput in field focused systems with a planar focusingsystem such as that shown in FIG. 11. For this system, many particlescan be imaged simultaneously. By using a wider field of view, somespatial resolution may be lost, but many applications do not requirediffraction limited resolution. Controllable velocity can make thistechnique extremely sensitive and also easier to implement. Pulsed flowcan also be used by taking images of particles/cells in batches: take apicture, then flush out the already imaged cells while replacing themwith a new batch of cells.

The statistical power of the cytometer of the present invention and thespatial resolving power of imaging are combined when additionallyimaging particles. This combination is very significant for numerousapplications where cell morphology and or localization of markers areimportant. Fluorescence in-situ hybridization (FISH), cancer screening,intracellular and membrane protein/drug localization and co-localizationare a few of the analyses that could benefit tremendously. Otherapplications such as industrial process monitoring or monitoring ofenvironmental samples can also benefit.

Another application for imaging in flow is the use of spatially barcodedparticles for multiplexed assaying. The microfluid method ensures thatthe particles align properly by using very small channel dimensions. Theacoustic focusing preferentially orients such particles in the fieldmaking it possible to use much larger channels that are not prone toclogging.

Methods for Monitoring Cell/Particle Kinetics in Field FocusedParticle/Cell Analysis Systems

Another embodiment of the present invention provides for cell monitoringor particle reaction kinetics by the imaging methods above but thesekinetics can also be monitored without imaging in field focused systemswith long transit times. The transit times can be adjusted to monitorwhatever process is of interest. The method lends itself very well totechniques that use light activated species such as caged fluorophors orions, photoactivated GFP or photoactivated ATP or GTP. Such techniquesare absent in flow cytometry due to the need for longer analysis times.

Kinetics Quantification

The quantification of kinetic parameters such as antibody bindingconstants and enzyme substrate cleavage rates can be quantified usingin-line acoustic washing and analysis. By placing reactant of a knownconcentration in a laminar stream and acoustically transferring thereactive particles into the stream such that the time of exposure toeach other is known, one can determine kinetic parameters using datacollected from the beads in a cytometer. For example, a new antibody canbe tested by switching antigen coated beads stained with fluorescentantibody with known constants into a stream containing a knownconcentration of the new antibody. The fluorescence of the beadsrelative to controls indicate the new antibody's ability to displace theknown antibody. The time of interaction can be varied with flow rate. Ifthe constants to be measured are longer lived, starting the reaction byprediluting with reagent and measuring fluorescence over the course ofanalyzing the whole sample is another alternative.

Microsphere beads are used for a myriad of applications in samplepreparation and purification. Among the most common are nucleic acidseparation, protein fractionation and affinity purification, cellisolation and cell expansion. Beads are generally separated from samplemedia by centrifugation or magnetic means. The microsphere beads canalso be separated by acoustic means and are typically denser and lesscompressible than most biological materials. For these beads, acousticwashing with a fluid stream of high enough acoustic contrast to largelyexclude sample materials while allowing central focus of the beadsallows execution of protocols otherwise employing magnetic means andcentrifugation. For many protocols, this allows cheaper non-magneticbeads to be used. It also provides for automation of steps that aretypically carried out in sample tubes.

Magnetic and acoustic forces can also be used in tandem, allowingternary separations of magnetic and acoustic beads or magnetically andacoustically labeled cells. Of course, magnetic beads can be used in aconventional manner and then be further processed or analyzed usingacoustic sample prep and or an acoustic cytometer. This combination canbe quite powerful as magnetic forces generally excel at quickly andconveniently separating targets from concentrated samples while acousticcytometry excels at quickly processing dilute samples. Multiplexedmagnetic bead arrays are a prime example of this where the convenienceof tube preparation is combined with the power of acoustic cytometryanalysis.

Example Negative Contrast Particles Nucleic Acid Isolation

Negative contrast particles modified for nucleic acid capture areincubated with a lysed sample and flowed through the acoustic separatorwhere they are forced to the outside walls. They are then washed withthe acoustic field on and resuspended with the field off. Washing inthis way can be repeated as many times as is desired. If the particlesare of low enough acoustic contrast, they can also be washed withisopropanol and ethanol as required. Nucleic acid elution can beperformed with the appropriate buffer and particles can be removed bysimply turning on the acoustic field. Alternatively, further reagentscan be added for nucleic acid amplification directly in the chamber, asthe required thermal control if implemented.

Example Microbe Isolation from Blood

The low level of microbes found in blood during sepsis poses manychallenges to sample prep for concentration and isolation of thepathogen. Acoustic washing can be implemented in ways that solve many ofthese challenges. By flowing clean media down the center and flowingcontaminated sample around the clean central core, smaller microbes canbe effectively excluded from collection in the central core. The centralcollector removes blood cells and the outer collector, which at thispoint contains mostly platelets and the contaminating microbes, proceedsto a second separation in which a core dense enough to exclude plateletsbut not most microbes is used to collect and concentrate microbes. Thecollected sample can then be concentrated further or be sent to analysisand/or be cultured.

Electroporation

The possibility of washing cells into a medium for lysis or permeatingmembranes is possible using acoustic washing. This idea can also beapplied to electroporation whereby cells are acoustically focused into areagent loaded stream and are flowed through an electric field thatpermeates the cell membrane and allows reagent entry into the cell. Thefield can also be used for other reactions like electroluminescence.

The single line focusing possible in a line-driven system allows precisecontrol of the field parameters that each individual cell is exposed to.It also enables post electroporation analysis and sorting. For example,a host of different cell types can be processed and then sorted on thebasis of cell surface markers and cultured and or analyzed for theeffect as single populations. Electroporation can also be performedwithout acoustic washing but may be preferable for conservation ofreagents or limiting cell exposure to reagents.

High Resolution Continuous Field Flow Fractionation Using Pre-Focusing

According to one embodiment of the present invention acoustic fields areused to separate particles based upon one or more characteristics of theparticles such as size, acoustic properties or a combination of both.The uniformity of separation conditions with respect to each particlecontributes to the precision of the separation efficiency. The abilityto separate into discrete populations becomes compromised if a particleflows more slowly than another or if the particle is exposed to adifferent gradient field than another. Referring now to FIG. 25,focusing particles in acoustic capillaries to form a single file line isillustrated. The method can be used to provide uniform distance andacoustic field exposure during a particle separation by first passingparticles in a sample into channel 2503. Particles within the sample aremoved to first acoustic focuser 2505 which focuses them in single fileline 2509 with first transducer 2507. An initial concentration can beadjusted to minimize aggregate formation in the acoustic field andinsure minimal particle to particle interaction. The line of particlescan subsequently be fed into acoustic separator 2513 equipped withtransducer 2512 and multiple exit bins 2519 a, 2519 b, 2519 c forseparation and collection. The position of line of particles 2509 can beadjusted as it enters the separator portion of acoustic separator 2513by drawing fluid away or otherwise removing fluid through, for exampleside channel 2511. Both flow rate and power can be adjusted toaccomplish the desired separation. If several fractions are desired, thecollector portion of the channel can be constructed in layers or bins toextract different fluid lamina. This layered construction can also aidin automated operation of the separations by reducing the need to adjustfor parameters that might affect separations such as viscosity. If, forexample, the viscosity changes in a particular separation due to forexample, temperature change, a particular desired fraction might end upin a different bin than expected, but it can then simply be collectedfrom that bin. Particles according to this embodiment can have acoefficient of variation improved by >40% or even >80%.

The system and method of the present invention holds particular utilityfor separation of microspheres that tend to be more poly-disperse astheir manufactured size increases beyond about 3 microns. It is notuncommon for even relatively uniform size standards to have coefficientsof variation above 10%. This corresponds to a standard deviation of 0.6microns for a 6.0 micron particle. Resolution of the separation ofpopulations within this variation can be very fine when the particlesare well separated such that they do not interact with each other. Giventhat each particle has similar density and compressibility, the acousticradiation force is proportional to the volume of the particle.Therefore, the force on a 6.2 micron particle is about 10% greater thanon a 6.0 micron particle while the drag force is only about 3.2%greater. This means that in a uniform acoustic field, if the 6.0 micronparticle is forced by the acoustic field to move 1 mm in a fluid, the6.2 micron particle will move about 1.08 mm. This is more than enoughmovement to separate these particles into different bins. If by usingthis process standard deviation for collected fractions in the aboveexample is reduced to 0.3 microns this represents a 50% improvement.Reducing it to 0.1 microns represents about an 83% improvement.

Further Examples

The invention is further illustrated by the following non-limitingexamples.

Probes that Particularly Benefit from Longer Transit Time

Probes useful in accordance with the present invention and that areuniquely enabled by the present invention, include but are not limitedto the following:

Dimmer Labels

Extinction coefficient less than 25,000 cm⁻M⁻¹ e.g. Alexa 405 and 430and or quantum efficiency less than 25% including but not limited toruthenium, and Cy3.

Photobleach susceptible or triplet state prone dyes.

Lower Laser Power

Dyes that suffer from photobleaching including but not limited to bluefluorescent protein or triplet state quenching including but not limitedto PerCP from medium power laser spots (>10,000 W/cm²).

For ultra-high power laser (>50,000 W/cm², as often used in stream inair sorters). This utility expands to include most dyes includingPhycoerythrin and fluorescein.

Longer Lifetime (Continuous Wave Excitation)

Lifetimes greater than 10 nanoseconds including Q-dots, Q-dot tandems,lanthanides, lanthanide tandem dyes, transition metal ligand complexesand phosphor particles. The longer transit time allows more cycles ofexcitation and emission, resulting in more overall photons emitted.

Longer Lifetime (Pulsed or Modulated Excitation)

The same class of probes as for longer lifetime CW excitation benefitbut there is particular utility for luminescent probes including but notlimited to lanthanides/lanthanide tandems and phosphors that havelifetimes in excess of the transit time in conventional cytometry (>10microseconds).

Bioluminescent, Chemiluminescent

Any probe that produces light from a chemical process (includingphotoactivated processes) that takes longer to emit the majority ofphotons in a conventional cytometry transit time (>10 microseconds)including but not limited to luciferin/luciferase, Ca⁺²/aequorin.

Radioactive Probes/Scintillation

For extremely slow transit >100 milliseconds per particle. These typesof particles are not analyzed in cytometry (even slow flow) due to thelong integration times necessary to gather enough photons.

Probes Resistant to Photobleaching with High Laser Power

Using very high laser power (>50,000/cm²) combined with photobleachingresistant probes including but not limited to dye loaded nanospheres,Q-dots® or C-dots® and very tight spatial filtering (as in confocalmicroscopy) high signal to noise ratio can be achieved. Long transittimes of greater than 10 microseconds combined with a very tightacoustically focused “core diameter”, the spatial filtering achievessuperior signal to noise.

Dimmer Labels

Dimmer labels, i.e. those having low extinction coefficients and or lowquantum yields will yield more photons with longer excitation times.Some dyes may have particular utility in certain applications includingbut not limited to UV excited dyes for multi-color analysis includingbut not limited to Alexa 405 and 430 or relatively dim long Stokes shiftdyes including but not limited to APC-C7. Other dyes may offer moreopportunity for developing assaying that normally use brighter labelsincluding but not limited to using dimmer tandem dyes vs. phycoerythrintandem dyes.

Also included in the category of dimmer labels are naturally occurringfluorescent species including but not limited to NAD(P)H. Someapplications for monitoring of such low quantum yield species and slowertransit time systems can make this easier with higher sensitivity andmore importantly, greater fluorescence resolution between cells orparticles.

Less Photostable Dyes

Lower laser power and longer transit time can increase the overalloutput such that dyes, including but not limited to fluorescent proteinor BFP could become more useful in flow.

Probes Prone to Non-Radiative State Excitation

Fluorophores as diverse as Rhodamine Atto532 and green fluorescentprotein or GFP can give off many more fluorescent photons beforedestruction when allowed to relax from long lived triplet statesfollowing intense laser pulses. There is greater emission for pulseswith longer dead time corresponding to the dark time estimated fortriplet states (˜1 microsecond). This emission is also significantlygreater than for the equivalent power of continuous wave excitation. Inconventional flow, one could not use a system with such long dead timesbetween pulses, particularly for probes requiring longer relaxationtimes such as PerCP (˜7 microsecond triplet state lifetime). The presentinvention allows for these longer relaxation times.

Quantum Dots

With quantum dots, their long fluorescence lifetimes reduce the amountof light they can give off when their excitation time is limited. In alonger transit, field focused system however, as in the presentinvention this limitation does not exist. The long transit times canelicit very bright signals even from just a few Q dots. Another problemwith Q dots is that they are made using toxic materials creating largevolumes of potentially hazardous waste. With the present invention,fewer Q dots can be utilized and waste volumes in field focused systemsare typically ˜100-1000 times smaller.

Luminescent Probes

In general, any long lifetime probe will not emit photons as quickly asa shorter lifetime probe so the performance of nearly all such probescan be tremendously improved with longer transit times as more photonsper label can be emitted. Also, lanthanides can be loaded at very highconcentrations in nanoparticles due to the fact that they do notself-quench easily. This allows particle tags to be much brighter thantags in solution.

Lanthanides have fluorescent lifetimes on the order of microseconds tomilliseconds with the most common europium chelates having a lifetimearound 0.7 milliseconds. Such probes are used for extremely sensitiveassaying in which background fluorescence is gated out in time from theluminescent signal by pulsing the light source and waiting to collectlight the background fluorescence has decayed. This type of luminescentassaying are useful in the field focused, long transit time system ofthe present invention. Tandem lanthanide fluorophores or assaying thatuse lanthanide based energy transfer to conventional fluorophores suchas the europium/allophycocyanin based TRACE™ system (Perkin-Elmer,Waltham, Mass.) and the terbium based Lanthascreen™ (Invitrogen,Carlsbad, Calif.) can also be implemented effectively in the presentinvention. The lifetimes are shorter when energy transfer to the otherfluorophore is possible. The lifetimes are much too long to be practicalin conventional flow cytometry.

Medium switching can be applied to luminescent reactions for whichexposure to luminescence reagents is controlled in time. In addition,serial or parallel reactions designed for permeation or lysis ofmembranes in order to facilitate diffusion of chemi or bioluminescentspecies can be implemented with precise timing and nearly equivalentexposure of cells to reagents. For example, cells expressing luciferasecan be transferred into a medium containing both lysing/permeationreagent and luciferin. As the membrane becomes permeable to theluciferin substrate the cell will begin luminesceing.

This process can of course be extended to other procedures using cellpermeation such as gene transfection or cell loading of other membraneimpermeant molecules/constructs. This in-line permeation can becarefully controlled with regards to time exposure of cells to lysisreagents by transferring cells in and out of the reagents sequentially.

Absorptive Dyes/Axial Light/Less Absorptive Probes

Non-fluorescent absorptive dyes are used very commonly in microscopy butnot in flow cytometry due to small signal to background. With increasedtransit time, integration is possible such that the signal to noise canbe greatly increased. Additionally, advances in high speed linear arraydetectors make it possible to increase signal by spatial isolation ofthe axial (approximately 0 degrees relative to the excitation source)light loss. Such arrays can scan fast enough to suit a slow transit timesystem and can give information regarding not only axial light loss butseveral angles of light scatter.

With a slower transit time system, bead arrays using different color andconcentration of absorptive dyes can be made practical for multiplexassaying. For multiplexing with more than one color, two lasers ofdifferent color are required such that differential color absorption canbe observed. If the lasers are collocated, the detectors need to be madecolor sensitive unless the excitation is separated in time. Oneadvantage of such arrays is that the absorptive dyes do not interferesignificantly with fluorescence tagging. Alternatively, one laser can beused if absorption is combined with another parameter such asfluorescence. Still another embodiment uses absorptive dyes with a wideband excitation source such as an LED and at least two color sensitivedetectors.

With much slower linear velocities (cells can even be stoppedmomentarily) imaging in flow for Pap smears becomes much easier suchthat both morphology and the information from absorptive dyes can becollected on a field focused system.

Radioactive Tracers

Radioactive labels are useful in the present invention due to the needfor long exposure times. The exposure required is on the order ofseconds. In particular, pharmaceutical screening assaying that use smallmolecule species or other species where a fluorescent tag mightinterfere with the specific action of the pharmaceutical candidate beingtested can benefit from the present invention.

Bioluminescent and Chemiluminescent Probes

These probes can be extremely sensitive as their signal is createdwithout background producing excitation light. The signal from suchprobes is integrated over times periods from 10 microseconds to 10seconds or more, much longer than conventional flow transit times.

Raman Scattering Probes

The field-focused systems of the present invention allow signalintegration and averaging of noise such that detection of Raman signalsis possible.

Example 2

Acoustic washing of the sample can eliminate most or nearly allcentrifugation steps in flow assaying (or other types of assaying thatwould benefit from sample washing prior to analysis). This not onlysaves tremendous amounts of technician time, but it also automates atedious process that is prone to operator error particularly due tofatigue. It also reduces exposure of operator to potentiallybio-hazardous materials.

Adjustable Concentrator/Washer

Acoustic concentration and washing can replace centrifugation operationsfor many assaying methods. It has many advantages including extremelyclean and gentle separation and reduced operator variation. In additionit presents new opportunities for sample processing that cannot beachieved in conventional centrifugation. By adjusting the concentrationratios used in an acoustic washer, one can chose the outputconcentration of a sample. If for example, the sample's initialconcentration is 105 particles per milliliter and the operator desires afinal concentration of 106 particles per milliliter, the operator canselect flow rates for the sample and collection channels that achieve a10 fold concentration. This process can be automated such that the userneed only enter a concentration factor. It can be further automated byadding a spectrometer on or off-line from the separator that determinesinitial sample concentration based on light scatter and calculates thenecessary flow rates to achieve a concentration given by the operator.If a sample is too dilute for the separator to accomplish the desiredconcentration, it can also perform several concentrations in series. Iffor example the starting concentration is 103 particles per milliliter,the 10 fold concentration can be performed 3 times to achieve thedesired concentration.

Immunophenotyping Wash

Depending on the protocol used, sample prep may include just oneseparation with one device or it might include a series of steps toautomate a more complex protocol. FIG. 8 is a schematic of oneembodiment of the present invention. In an automated system, cells andlabels are added together and incubated initially. Then various stepsincluding washing, lysis, fixation and concentration can all beaccomplished using acoustic modules without centrifugation. For example,a first step includes the transfer of blood cells from serum toeliminate serum proteins and the wash medium can contain red cell lysingreagents. The next step includes transferring the remaining cells into aquench medium to stop lysis. This medium could contain stainingantibodies or the cells could be concentrated into an incubation chamberwhere antibodies are added. After incubation cells would then be sent toanalysis where they are washed in-line to eliminate background fromunbound antibodies.

It is possible to add additional processes and or washing modules inorder to automate further steps, e.g. adding more reagents, and/orprovide for extra washes if necessary. The acoustic cytometer is coupledto the detector and can also be fitted with an in-line medium switcherif desired.

While FIG. 8 illustrates a serial process using more than one separator,it is also possible to do “parallel” media changes in which more thanone wash or reaction medium is flowed into a single separator andparticles or cells pass sequentially through the media lamina as theymove toward the center (FIG. 12). This concept can be extended toinclude a continuous gradient type of fractionation in which severalfluids of incremental contrast are simultaneously injected into theseparator. One can envision several complex protocols automated byadding more and more serial or parallel media changes or combinationsthereof.

FIG. 12 illustrates a schematic of parallel medium switching device.Multiple media can be used in laminar layers. Sample 1205 is added tocapillary 1201. Sample 1205 contains a first particle 1204 and a secondparticle 1202. A second medium is added to capillary 1201. A thirdmedium 1209 is added to capillary 1201. A line drive 1203 inducesacoustic wave and first fluid, second fluid, third fluid, first particleand second particle are acoustically focused/reoriented based upon theacoustic contrast of each. Acoustically focused particles flow out ofthe capillary 1213. The third fluid is preferably introduced into achannel, the third fluid having a third acoustic contrast relative tothe first fluid and the second fluid. The third fluid may containparticles, and the third fluid preferably moves in a third laminar flowstream. The third fluid can have an acoustic contrast that is greaterthan, lesser than, or the same as the acoustic contrast of the secondfluid. The third stream can also be acoustically reoriented based uponthe acoustic contrast of the third fluid. A portion of particles thatmay be in the first fluid can be acoustically focused from the firstfluid to the third fluid. This portion of particles preferably passesthrough the second fluid, wherein the second fluid is preferably areagent stream. A portion of particles may also be acoustically focusedfrom the second fluid to the third fluid.

Immunophenotyping Panels

In clinical immunophenotyping laboratories, assaying is often done on asingle patient's blood in order to classify a particular disorder. Theamount of assaying can be reduced by increasing the number of markersthat can be assayed at once. Assaying is mostly performed with no morethan 4 antibodies because of overlapping spectra for fluorescent tags.Controls for compensation in which each assaying is run without one ofthe four antibodies greatly increase the amount of assaying that must beperformed and add a huge burden in terms of technician time, reagentconsumption and analysis time. Performing the current panels withoutneed for compensation promises to greatly streamline the process andperforming larger compensation free panels of, for example 6 or moreantibodies at once, can reduce assaying significantly.

Compensation is simpler for assaying with fewer colors but they can alsobenefit from a compensation free panel of antibodies. A very commonexample is a panel of anti-CD45, CD4, and CD8 antibodies which is usedfor CD4 positive enumeration of T-cells in AIDS progression monitoring.CD3 is often added or substituted in this panel to aid withidentification of T-cells.

Table 1 below is an example list of assaying for four colors done fornew patient classification of leukemia/lymphoma. This screen is used fordiagnosis of new patients where the disease classification is not known.The four cell markers are listed on the left and the utility of assayingis listed at right. Typically analysis is done on a blue (488 nm) andred (635 nm) laser cytometer with each antibody having a differentfluorochrome. A very common combination is FITC, PE, PE-Cy5® and APC. Inan acoustic cytometer equipped with long lifetime analysis capabilities,one or more probes can be replaced with a long lifetime probe for whichoverlapping spectral signal can be subtracted based on temporalmeasurements. Long transit time can also be used to make up for lostphotons if fluorescence filters are narrowed to prevent overlappingspectra. In a system equipped with a violet laser and a blue channelthat is not otherwise used to detect a short-lifetime probe,auto-compensation for autofluorescence can be done on a cell by cellbasis.

TABLE 1 CD3 This antibody combination is designed to give adifferential. CD14 CD45 vs SSC is used to define lymphocytes, monocytesand HLADr granulocytes. CD14 further defines monocytes while CD3 givesCD45 T-cells and HLADr identifies B-cells and NK cells. In bone marrow,progenitor cells are CD4 dim HLADr+ and erythroid cells or platelets areCD45 negative. CD7 This combination is used for three reasons. 1) NormalT-cells CD13 express CD2 and CD7, which are often expressed at abnormalCD2 levels by malignant T-cells. NK cell malignancy is usually CD19CD7+CD2−. 2) CD2+CD7+CD13+ cells represent aberrant co expression of themyeloid antigen, CD13, on acute T lineage lymphocytic leukemias andlymphoproliferative disorders. 3) Co expression of CD2 or CD7 or CD13with CD19 defines aberrant expression of these markers on B lineagemalignancy. Finally, CD19 is often co-expressed on CD13 AML-FAB/M2 witha t(8, 21) translocation. CD5 This combination is designed to resolveB-lineage Lambda lymphoproliferative disorders. Clonal excess of kappaor lambda CD19 on CD19 positive cells or CD19CD5 positive cells areexplicitly Kappa defined. CD20 This combination is used to furthercharacterize B-lymphopro- CD11c liferative disorders and to define thedegree of maturity of acute CD22 B-lineage leukemias. In addition, hairycell leukemia can be CD25 classified by its unique high expression ofCD11c. Aberrant expression of the T-cell marker CD25 on B-cells isdiagnostic for lymphoproliferative diseases when it is expressed. CD5This antibody combination is designed to resolve the cells within CD19the maturation of both T and B-cell lineages. The earliest T-cells CD10are CD5+CD10+CD34+, which differentiate into CD5+CD10+ CD34 by losingCD34 and finally into mature T-cells that express only CD5. Thiscombination can be used to evaluate the maturity of a T lineage acuteleukemia. In a like manner, the maturation of the two distinct B-celllineages: CD19+CD5− and CD19+CD5+ can be defined. The earliest B-cellsco express both CD34 and CD10. As maturation occurs, they lose CD34,then CD10 to become mature B-cells. CD15 This combination is used todefine aberrant antibody expression CD56 on hematopoietic malignancies.CD56 is expressed early on CD19 progenitor cells (CD34+CD56+) that mayalso co-express CD15. CD34 We have shown that in acute leukemia,co-expression of CD15, CD56 and CD34 is associated with the t(8, 21)translocation, which results in a very bad prognosis. CD15 is alsoexpressed on granulocytes and some B-cells.

Table 2 below illustrates examples of assaying six colorleukemia/lymphoma cells that utilize six labels to reduce the amount ofassaying that must be run. Each assaying is numbered on the left, thetop column is the fluorochrome used for each antibody and thespecificity of each antibody is listed left to right underneath itsrespective fluorochrome label. In this table there is significantspectral overlap. Again by replacing fluorochromes with a long-lifetimereagents and narrow band reagents, minimal compensation antibody panelsare possible.

TABLE 2 FITC PE PerCP-CY5.5 PE-CY7 APC APC-CY7 1  CD7 CD4 CD2 CD8 CD3CD45 1. Kappa Lambda CD5 CD10 CD34 CD19 2. CD38 CD11c CD22 CD19 CD23CD20 3. CD57 CD56 CD33 CD8 CD161 CD3 4. CD11b CD13 CD33 HLADr CD34 CD455. CD71 CD32 CD41a CD16 CD64 CD45

Table 3 below shows an example of labels that accomplish compensationminimized results that do not require compensation controls. Theinstrument uses 405 nm and 635 nm pulsed diode lasers.

TABLE 3 Qdot ®545 Qdot ®800 EuropiumDEADIT PerCP APC AlexaFluor ®405 1 CD7 CD4 CD2 CD8 CD3 CD45 1. Kappa Lambda CD5 CD10 CD34 CD19 2. CD38CD11c CD22 CD19 CD23 CD20 3. CD57 CD56 CD33 CD8 CD161 CD3 4. CD11b CD13CD33 HLADr CD34 CD45 5. CD71 CD32 CD41a CD16 CD64 CD45

Temperature

Temperature can also affect specific gravity and therefore acousticcontrast. This feature can be manipulated by pre-cooling and/orpre-heating one or more of the input streams or by heating or coolingdifferent parts of the separator so as to create a temperature gradientin the fluid stream.

Example 3 In-Line Red Cell/Cell Lysis

By incorporating a rapid red cell lysis reagent into the central washstream, it is possible to lyse red cells in-line in a flowing separator.After lysis, the unlysed white cells can be quickly transferred to aquenching buffer in a subsequent separator. This operation can beperformed in seconds, minimizing damage or loss of white cells. Thesecond operation can also be used to exclude debris including lysed redcell “ghosts” that have decreased acoustic contrast resulting from thelysis process.

Concentration of Analytes/White Cells to Decrease Labels or Time

Often, staining of white blood cells for immunophenotyping is done in asmall volume of blood prior to lysis. Alternatively, staining can bedone after lysis but the sample volume and number of white cells must becarefully controlled in order to insure the proper immune-reaction. Theacoustic wash system can be used to concentrate target cells orparticles to a small volume for proper immunostaining. This feature isparticularly valuable for samples with a low concentration of targetcells as it allows a smaller staining volume and therefore lessantibody. For example, such a system can be used to decrease the cost ofassaying in CD 4+ T cell counting for AIDS progression monitoring. It isalso valuable for removal of native serum antibodies that mightinterfere with proper white cell staining, particularly when coupledwith acoustic washing. It is also valuable for removal of native serumantibodies that might interfere with proper white blood cell staining,particularly when coupled with acoustic washing.

Acoustic No Lysis Protocols

Immunophenotyping in blood is sometimes performed without red cell lysisby triggering detection on fluorescence signals rather than scattersignals. In these protocols, whole blood is stained with appropriateantibody and fed into a cytometer without lysis, in some cases withvirtually no dilution. An acoustic cytometer according to one embodimentof the present invention is capable of performing this type of assayingwith higher throughput of between approximately 100-500 μl of wholeblood per minute since the blood cells can be concentrated into acentral core with very little interstitial space. The white blood cellsin normal patients usually make up less than 1% of the total number ofcells in whole blood so coincidence of white cells in the dense bloodcore is rare. Hydrodynamic focusing cannot form such a solid core andcan therefore not pass as many cells through a given cross sectionalarea. An additional advantage to formation of such a core is that allcells in the core travel at the same speed allowing for uniform transittimes through a laser spot. The no lysis protocol can be furtherimproved by adding an acoustic wash step that transfers the blood cellsaway from free antibody and into clean buffer. This reduces fluorescentbackground and increases sensitivity. In addition, the clean buffer canbe adjusted to have an index of refraction that closely matches thecell's index. This has the effect of reducing scattering of the laser bythe cell core.

FIG. 13 is an illustration for stream switching of unlysed whole bloodaccording to one embodiment of the present invention. Because of theirrelative low numbers white cells maintain separation in the rope likestructure of focused blood. Capillary 1302 receives blood sample 1309and wash buffer 1307 at different spatial locations of the capillary1302. Red blood cell 1303 and white blood cell 1305 are acousticallyfocused and sample 1309 and wash buffer 1307 are acoustically reorientedupon activation of the transducer 1304 which produces an acoustic waveof a user defined mode within the capillary 1307. Cells are acousticallyfocused based upon their acoustic contrast.

Example 4 Urinalysis

Analysis of particles/cells in urine is a very common test used toscreen and diagnose many conditions including urinary tract infectionsand urinary system cancers. Most commonly, particles/cells in urine arecentrifuged to concentrate them and then they are examined using amicroscope slide. This is time consuming, labor intensive and subject tooperator error as well as error from the effects urine can have oncellular constituents.

Urine is a destructive environment for cells as it can havenon-physiological osmotic pressures and pH as well as toxic metabolites.These conditions dictate a minimal post-collection delay for examinationto avoid excessive degradation of cellular targets. This exposure can beminimized in an acoustic washing system by transferring the urine samplecells and particulates immediately into a cell friendly wash solution.The concentrating effect of the system is particularly well suited tourine processing where the cells and particulates tend to be of lowconcentration. Concentrated and washed fractions can be processedfurther as needed for a particular assaying. Reagents can be added,cells can be sent to culture or genetic analysis and/or an in-lineanalysis step can be added.

As urine density can vary widely, the wash fluid should be denser thanthe maximum density expected for the patient population tested (orcompressibility should be adjusted accordingly). Urine sometimescontains mineral or other crystals that can be highly dense and a serialfraction that isolates these components with a very high density washstream followed by a second, less dense wash to capture other componentsmight be desirable in some cases.

Example 5 Coulter Volume Sensing/Electrical Measurements forCells/Particles from Uncalibrated Solutions/Buffers

By acoustically transferring cells or particles into a solution that iscalibrated for conductivity, particle counting can be done in linewithout centrifugation or dilution. This is of particular value fordilute samples where such manipulation by centrifuge may be difficult.It also enables automated continuous monitoring of some process.Monitoring particles in municipal water supplies is a good example asparticulates are a very small volume fraction and continuous watermonitoring might be desirable. One can envision a continuouslyoperational system that might trigger more analysis as particles ofcertain size and characteristic increase. After triggering, for example,DNA dyes for revealing if the particles might be a biological threat canbe added to the washing core and the particles would be sent tocytometric analysis.

Acoustic focusing by itself provides further advantage to pore basedelectrical measurements as it ensures that particles to be analyzed passthrough the measuring orifice in its center (FIG. 14). This makesmeasurements more precise and allows for smaller particles to beanalyzed in larger less clog prone pores. This way, an instrument cancover a wider range of particle sizes without changing pore size as isthe common practice.

FIG. 14 is a schematic example of an acoustic stream switching particlecounting device 1400. The design allows for in-line analysis of samples1405 in unknown or unusable conductivity buffer 1403. Even withoutstream switching the acoustic positioning of particles 1409 improvesperformance over a broader range of particle sizes for a giveninstrument pore size 1419. Transducer 1407 provides an acoustic wave tothe flow cell. Particles 1409 are acoustically focused to buffer 1403.Sample medium is discarded at 1411. Electronics detection 1417 detectssignals at electrodes 1415 after particles pass from the secondtransducer 1413 to the detection pore 1419.

Example 6 Bead Based Reactions, Purifications and Assays

Polymer beads, including but not limited to polystyrene beads, are veryuseful in embodiments of the present invention. Having a somewhatsimilar (slightly higher) positive acoustic contrast to cells, they canbe manipulated in similar fashion. Being hardier than cells however,they can be subjected to harsher environments that might damage ordisrupt cells. For example, high salt environments can be used in beadbased immunoassaying to reduce non-specific antibody binding. This canbe done with cells as well but salinity and or exposure time must belimited if membrane integrity is required.

Beads of many different materials can be manipulated differentlyaccording to their acoustic properties. High specific gravity/lowcompressibility beads including but not limited to silica or ceramicbeads can be acoustically focused through a high specific gravitycentral core that excludes the cellular debris in a lysis protocol.Negative acoustic contrast particles, e.g. silicon rubber, can bemanipulated in opposite fashion such that they move to the outside wallof the capillary through a low specific gravity buffer, leaving cellulardebris and uncaptured protein/nucleic acid behind in the center see(FIG. 15).

FIG. 15 is a schematic example of separation of negative contrastcarrier particles 1505 from a core of blood sample 1503 and 1511. Thenegative contrast carrier particles 1505 leave the core 1502 and passthrough clean buffer 1503 before approaching the capillary walls 1501. Atransducer 1507 induces an acoustic wave that acoustically focuses thenegative contrast carrier particle to the capillary walls and focusesthe blood all to the center. Other acoustic modes exist that make itpossible to invert the image. Blood cells can be driven to the walls andnegative contrast carrier particles to the central axis

Example 7 Immunoassaying

Bead based sandwich immunoassaying benefit from acoustic wash in thesame manner as immunostaining of cell surface markers. Centrifugationsteps to eliminate excess antigen and reporter antibody are replacedwith rapid in-line acoustic washing. The washed product is assayed in aconventional manner (e.g. bulk fluorescence, plate readers) or it is canbe coupled to flow cytometry analysis, particularly if multiplexingusing soluble bead arrays is desired. An apparatus useful to processsamples for a plate reader provides all of the advantages of bead basedassaying (inexpensive volume manufacture, better mixing and kinetics)with the existing infrastructure and easy calibration of plate readingassaying. Even enzyme linked assaying is carried out with the finalamplification step being accelerated by active mixing with the beads.

Competitive immunoassaying can be performed very quickly by flowing theanalyte in the center stream and pushing beads pre-bound withfluorescent antigen into the stream. As the fluorescent antigen isdisplaced by native antigen from the analyte medium, the change influorescence of both the beads and the background stream can bemonitored in real time. The beads become dimmer and the backgroundbecomes brighter as the fluorescent antigen is displaced and diffusesout into the stream (see FIG. 16). As flow rate in an acoustic cytometeris adjustable, the flow rate should be adjusted to match the kinetics ofthe assaying such that close to maximum signal is achieved at thedetection point. By moving beads into the analyte stream andacoustically concentrating them therein, these problems of diluting theanalyte, limiting assaying sensitivity, and long analysis time areeliminated. In addition, individual bead detection allows multiplexingfor simultaneous detection of multiple analytes. Examples of suchmulti-analyte testing include but are not limited to blood donorscreening, and/or STD testing.

FIG. 16 illustrates a schematic example of multiplexed competitiveimmunoassaying in an acoustic wash system 1600.

Example 8 Staining of DNA/RNA Hybridized to Beads

Washing steps are eliminated for DNA/RNA prep and analysis as forprotein analysis. Conventional labeling strategies using biotin oranother linker are used with the final step being acoustic eliminationof the reporter label prior to analysis. In addition, for native DNA orDNA without a linker, intercolating dyes are added to the wash stream inorder to stain DNA hybridized to beads. Only double stranded DNA boundto beads are stained with this technique. This technique may beparticularly useful for unamplified analysis of nucleic acid fragments(such as micro-RNA, plasmids or enzyme digested/mechanically fragmentedgenomic DNA).

These nucleic acids are extracted from a sample by hybridizationcombined with attachment of a high acoustic contrast label. The processincludes hybridization of a probe with a linker including but notlimited to biotin, followed by for example binding of streptavidincoated particles with high acoustic contrast including but not limitedto silica, gold, or negative contrast silicone rubber. In this system,if multiplexing is desired, the probe itself may need to be coded insome readable fashion. Positive hits may only be recorded for signalsthat combined the coded fluorescence with signals from dyes intercolatedinto the hybridized nucleic acid.

Nucleic acids tests can be made more sensitive and specific if nucleicacid degrading enzymes are included in the transfer media (or a transfermedium in a previous step) and hybridized products are protected fromdegradation by protective modification of the DNA/RNA probes. In thisway any nucleic acid not hybridized (including that non-specificallybound to beads) can be enzymatically degraded.

Example 9 New Cellular Analysis Tools/Applications

Acoustic manipulation of cells lends itself well to non-adherent cellline or cells that have been harvested from adherent culture. Thiscombined with acoustic cell medium switching enables many new in-linemanipulation techniques that enable new assaying, gentler and quickerhandling of cells and lab automation.

Production of Fused Cell Lines

A critical step in the production of fused cell lines such as tumorcell/dendritic cells or B-cell hybridomas for antibody production isgetting the different cell types to contact each other prior to fusion.Line driven acoustic focusing in conjunction with optimization of sampleconcentration can be used to line up the different types of cells inseparate lines of optimal spacing. These separate lines can then bejoined by flowing them both into another focuser that then joins the twolines. Electric fields can then be used to fuse the cells either in flowor not. If desired this line can then be further analyzed or sortedprior to culture.

Referring now to FIG. 24, acoustic positioning of particles for fusionor reaction is illustrated. First sample 2401 containing a first cell orparticle type is adjusted to an optimal concentration for interactionwith a second particle type. The sample is pumped through first acousticfocuser 2402 driven by a PZT transducer 2404 and the particles areacoustically focused into a line 2408 with particles having a spacingaccording to the sample concentration. A second sample 2403 containing asecond particle type is similarly adjusted for concentration and thenpumped and focused into a line 2409 in the second acoustic focuser 2405driven by PZT transducer 2407. The flow from both samples is flowed intoa third acoustic focuser 2410 driven by PZT 2411 such that each focusedline of particles flows parallel to the other. When the acoustic fieldfrom the acoustic focuser 2410 is switched on, the two separate lines ofparticles focus to form a single line where particles from sample 1 andsample two can interact. Acoustic Bjerknes forces in acoustic focuser2410 act to bring close particles into contact. Downstream, afterparticles are in contact, the pass through an electric field producedusing electrodes 2413. The electric field acts to fuse cells and thefused cells 2412 may be collected for culture or sent to another processsuch as analysis or triggered sorting. This device is useful forproduction of fused cells such as antibody producing hybridoma cells andcan be applied to any process requiring interaction between suspendedparticles.

The method is not limited to fusing cell lines but can be appliedwherever close interaction of particle populations is desired. Anotherimportant example is fusing aqueous drops in oil. In this case, thecarrier medium is oil and the particles are the water droplets. Eachdrop population can be loaded with different reagents that interact uponfusion of the droplets. The droplets can also then be analyzed or sortedin flow. Other examples include exposing cells to beads with reactantsas in T-cell activating antigens or exposing macrophage or monocytes tobacteria or other particles for phagocytocis.

Another embodiment of the present invention comprises joining three ormore acoustically focused streams of particles in the same fashion byflowing them all into a single acoustic focuser where they are broughtinto close proximity be the acoustic field.

Still another embodiment of the present invention uses two acousticfocusers. The first focuser focuses the first particle population andfeeds the line of particles into the center of a second acousticfocuser. The second population of particles is then fed around the axialedges of the second focuser and is acoustically focused into the centerwhere they join the first line of particles.

Flow Through Affinity Harvesting of Cell Products

Affinity purification of cell products such as antibodies is typicallyaccomplished using columns. Bead based methods using centrifugation ormagnetic batch separation are also available. Acoustic separation can beused to accomplish this in a flow through fashion using affinity beads.For example, specific affinity beads such as those coated with anantigen of interest or protein A or G for capture of the Fc region ofantibodies would be incubated and mixed with spent medium or anti-serato capture antibodies. The beads can then be concentrated and collectedin a flow separator and washed if desired. The wash medium may beformulated to discourage non-specific binding, e.g. high salt. Thecollected beads are then exposed to conditions which disrupt thespecific binding after which they are again collected on a flow throughseparator where they can be recycled for the next purification. Ifdesired the specific binding disruption can be accomplished in flow ifminimal exposure to these conditions is desired. This is done by in-lineacoustic medium exchange with the dissociating medium. The dissociatedproduct is collected independently from the beads and processed asnecessary, e.g. ammonium sulfate precipitation for antibodies or otherproteins.

Harvesting of Cells or Particles

When the product to be harvested are the cells themselves, an acousticseparator can be used to concentrate and collect the particles with anaxial collector or if the concentration of cells is high enough it canaggregate the cells into a continuous flowing line or line of clumpsthat can be fed into a collection vessel where flow is slow enough toallow settling by gravity or removal by other means. This method wouldbe particularly useful for filterless continuous collection ofmicroalgae for biodiesel production.

Harvesting of Lysis Products from Cells or Particles

If the cell or particle must be broken or lysed to harvest the materialof interest, acoustic separators may be employed to separate lysisdebris from the material and may also be used in-line to initiate lysis.Microalgae lysates are a special case where the product of interest,algael oil is separated and focused to the outside of the capillary andcellular debris and residual water goes to the center. If an appropriatelysis fluid is used, a simultaneous algae collection, lysis and algaeoil step can be performed in which harvested algae are fed into onestream and lysis fluid fed into the other. Debris, lysis fluid andculture medium are collected in the center and oil is collected to theoutside.

Example 10 Radio Ligands/Drug Candidates

The ability to leave the original medium behind allows the combinationanalysis of long exposure time indicators such as radioactive ligands ordrug candidates with single cell analysis and sorting techniques. Forexample, a radio labeled drug candidate can be added to a single wellwith several different cell types. Cell types incorporating positive andnegative controls, including but not limited to cells from a parent linethat have not been modified to contain the receptor of interest and celllines with known activity. Relative cell size and granularity can beexamined and multiple color analysis can be used to extract manyparameters from each individual cell. Each cell type can be identifiedwith cell specific fluorescent antibody combinations or with fluorescentfusion proteins/gene reporters, including stably expressing lines. Awide variety of intrinsically fluorescent reporter proteins and reporterprotein systems that become stained with additional reagents may be usedin the present invention. Many other reporters can be used to indicatecell conditions including but not limited to growth phase, pH, lipidrelated toxicity, etc. Receptor expression levels and internal fusionprotein expression levels can be monitored, FRET interactions can betracked. In short, any fluorescent parameter that can be monitored byflow cytometry can be utilized in the present invention. The multiplexedcell sample is washed in-line acoustically leaving excess radio ligandsbehind. The cells are then analyzed individually using acoustic flowcytometry and the analysis is sorted as to individual cell populationsby fluorescence activated cell sorting (FACS). The collected populationis then radioassayed for the amount of drug that remains with eachpopulation FIG. 17. If desired the cells can be acoustically transferreddirectly to a scintillation medium. With an acoustic flow cytometer,this process of washing, analyzing and sorting can be accomplished infractions of a second, leaving little time for bound drug molecules todissociate from cells. This process is particularly useful for drugcandidates or other ligands that cannot be readily labeled withfluorescent or other large reporters without affecting activity.

FIG. 17 illustrates an example flow chart for high throughput/highcontent screening using acoustic medium switching. Cells or particles1701 are incubated with labels and or drug candidates of interest 1703after which they are sent to the acoustic focuser/stream switcher 1705where they are separated from excess drug/ligand. Optionally a differentreactant 1709 can be placed in the new medium such that cells/particlesinteract with it during acoustic separation. Additional acoustic switchsteps can be added in serial as in FIG. 8. Cells are then collected 1707for analysis and or sorting 1711. Unwanted cells/particles can be sentto waste 1713 while selected particles are sent to additional analysisor processes 1715. Some useful examples of such processes are listed in1717.

Of course, the acoustic washing process can be used with most anyreporter and is of particular utility for any application where theconcentration of ligand should be maintained up until just prior toanalysis. The process can also be extended to any ligand/drug candidatethat can be assayed after analysis. If, for example, a library ofproteins is synthesized with a sequence that allows fluorescentstaining, the staining can be done after washing and sorting todetermine how much was bound to the sorted population.

If only one cell population is used, sorting is not necessary, but datacollected from the single cell analysis is useful in determiningvirtually any other parameter that can be monitored by fluorescentacoustic cytometry, e.g. number of live/dead or apoptotic cells.

Example 11 Calcium Activation

Acoustic washing of the present invention can be used to simultaneouslysimplify and improve calcium activation assaying or other ion probeassaying in flow cytometry. Calcium activation studies are normally doneby preloading target cells with calcium sensitive reagents, washing awayexcess reagent and other media components that contribute to backgroundfluorescence, exposing the cells to a calcium activator or drug compoundunder test and monitoring the cells for changes in optical signal. Theassaying is often done quickly after the washing step in order to keepthe concentration of the reagent within the cell high. “No wash”assaying has been developed to improve precision by maintainingequilibrium between intracellular and extracellular reagents but otherreagents are used to reduce background such as probenecid which inhibitsactive transport of the reagent outside the cell or quencher dyes whichreduce the fluorescence of extracellular dyes.

With acoustic washing, reagents and fluorescent media can be rapidlyremoved just prior to analysis, eliminating the need for quenchers ortransport inhibitors. Washing need not be done prior to adding thecalcium sensitive reagent. For example, cells may be maintained in aculture medium if desired and minimizes the use of other reagents thatmight interfere with the activator or the cell response is minimized.The calcium activator or test compound can be added to the acoustic washsolution or it can be added just prior to the acoustic wash depending onthe users desired measuring time point. The reagents for use in anacoustically washed calcium activation assaying is then simply at leastone calcium sensitive reagent and an acoustic wash buffer engineered tohave an acoustic contrast higher than the cell sample medium. Examplesof calcium probes are the Indo series, Bis-Fura, Fura series andFuraRed™, Bis Fura, MagFura series, BTC, Calcium Green™, CalciumOrange™, Calcium Crimson™, Calcium 3™, Rhod™ series and X-rhod™ series,Magnesium Green™ and Oregon Green® BAPTA series. A second calciumindicator can be added to increase dynamic range measurements in a cellor a non-calcium dye can be added for reference. Combinations of dyeswith reciprocal changes in fluorescence upon calcium activation havealso been used to do ratiometric measurements with non-ratio-metricdyes, e.g. fluo-3 and Fura Red. Calcium indicators can be supplied in anumber of different forms including cell membrane permeant AM esters anddextran conjugates designed to block internal cellular sequestration.

One embodiment of the present invention comprises a method for measuringcellular calcium concentration in an acoustic particle analyzer. Thisembodiment preferably introduces a calcium sensitive reagent into apopulation of cells to be analyzed. The population of cells is thenmoved through a channel wherein the population is acoustically focusedin the channel. The population of cells is exposed to a reagent that mayor may not induce a cellular calcium response. The population of cellsis then preferably passed through an interrogation point and collectingsignal to determine calcium concentration in the cell. In thisembodiment, the population of cells can optionally be washed prior toanalysis and/or diluted prior to analysis. The flow rate of thisembodiment is preferably adjusted to achieve a desired time of analysisafter the exposure to the reagent that may or may not induce a calciumresponse.

The power of flow cytometry to distinguish individual cells makes theprospect of engineering different cell types with the intent ofsimultaneously testing them for drug candidates or other activators veryattractive. Each cell type can be engineered with different receptortypes or receptor expression levels and can be uniquely identified usingcharacteristic markers or other reporters. Different cell types can thenbe simultaneously mixed together and tested for response. Positive andnegative controls can also be combined with various cell types beingmonitored.

The process can also be implemented in-line such that the ligand or anadditional ligand(s) are serially injected into the core stream and thecells interact with the injected ligand. This is particularly useful forfast kinetic processes and can be combined with a kinetic analysistechnique such as calcium sensitive fluorescence dye response. If radiolabeled ligand is used, an additional in-line wash step might berequired to eliminate the free ligand before sorting. Alternatively, aparallel system can be used where the cells pass through a layer of theligand into the clean wash (see FIG. 12). In this system, interactionwith the ligand will only occur as the cell passes through the ligandlayer. Even if no radioligand is used, this medium switch method can beused to extract high information content as above in combination withcalcium response or other kinetic analysis. The ability to adjust flowrates as desired enables tuning of each assaying to reach analysis atthe desired time course. Kinetic response for a population of cells orbeads can also be monitored by ramping flow rate up or down such thatcells arrive at different times during the response curve.

A sensitivity problem for calcium response measurements lies in theability to analyze quickly enough and for long enough after the calciumflux inducing ligand is added to catch and integrate the peak responseof the cell population involved. The stimulant must be quickly mixedwith the sample during analysis and analyzed cells end up with verydifferent exposure times. With the acoustic media switching method ofthe present invention it is possible to precisely adjust flow rates suchthat cells arrive at analysis when desired and that they are monitoredlong enough to collect more signal. The method also insures that eachcell in the population is exposed for the same length of time to thesame concentration.

In addition to fluorescent parameters, any of these medium switchmethods can also be combined with acoustic flow cytometric imaging whichcan provide additional valuable spatial fluorescence/luminescenceinformation, morphology and spatially relevant absorbance information.The ability of the acoustic cytometer to drastically slow flow rates oreven stop for a triggered event allows for high resolution imaging andhigh resolution spectroscopy, owing to the ability to integratedetection light for longer.

If cell population data is more important than individual cell data,many of the assaying protocols above can be performed in systems withsimpler optics. Instead of probing single cells, a population can bemonitored just after processing in afluorescent/scintillation/luminescent plate reading device.Alternatively, reactions can be monitored inside the capillary bycollecting light at either end.

Example 12 Low Affinity Drug Candidates/Ligands

Low affinity fluorescent ligands can be used in higher concentration ascells can be transferred from the high fluorescent background of excessligand just before analysis in a time frame that does not allowsignificant dissociation. At high concentrations where non-specificbinding of low affinity ligands may interfere with accurate analysis,acoustic washing into high salt buffers helps to favor specificreactions while accelerating non-specific dissociation.

Example 13 Multiple Ligands/Compounds and Serial/Parallel Processes

Much of biology and chemistry depends on the interaction of severaldifferent species of molecules or ligands. Acoustic medium switchingprovides a rapid and convenient means to expose cells or particles to aseries of different compounds/ligands in rapid succession. This can bedone in series and/or in parallel. It can also be readily modified toinclude changes in reactants or reactant concentration if the system isequipped to inject different media in the core streams or lamina of thedevices.

Serial reactions can be performed for drug/ligand discovery in much thesame way as shown in FIG. 11.

This can also be done in a triggered fashion in order to save resources.If for example a drug target is identified as a hit for inhibiting cellactivity, it is then desirable to confirm the health and viability ofthe cells to exclude acute cytotoxicity of the compound. In this case,any “hits” can be diverted to a system performing viability testing.Alternatively of course, viability testing can be done simultaneously ifa separately distinguishable health/viability marker is used.

Example 14 Enzyme Reactions

Enzymes are a special class of molecules that can be placed either inthe original sample or in the medium that cells or particles areswitched into. If cells or particles are acoustically transferred awayfrom the enzyme, this will serve to stop an enzymatic reaction. If theyare transferred into a medium containing enzyme, this will start thereaction. Enzymes are used for many protocols in sample prep, includingbut not limited to degradation of cell walls or unwanted nucleic acid.They are also used to detect or amplify detection of specific moleculesincluding but not limited to fusion protein labeling or ELISA. They arealso used as drug screening tools, e.g. candidates are monitored fortheir ability to block or inhibit the activity of specific enzymes. Allof these applications are implemented in acoustic medium switching. Inthe last application for example, beads coated with fluorescent or FRETor quenched fluorescence enzyme substrate can be switched to a streamcontaining enzyme that was treated with a drug candidate. Thefluorescence of the beads and the diffusing fluorescent substratecleaved by the enzyme can be monitored much in the same way as describedpreviously for the competitive immunoassaying (FIG. 16).

Example 15 Acoustic Medium Exchange for Bio/Chemical Synthesis,Bioreactors and Other Industrial Processes

Bio/Chemical Synthesis

Methods using cells or beads for sequential processes that requiremedium exchange can benefit greatly from the speed and automationpossibilities of acoustically transferring particles across differentmedia. Compounds synthesized on the surface of beads for example, can betransferred from medium to medium through the synthesis protocol.Another example is transfer of cells through media containing differentgrowth factors designed to promote expression of a protein of interestin a sequential protocol.

Bioreactors

Ultrasonic concentration can be achieved, including but not limited to,antibody production using hybridomas and flocculation of microalgae forbiodiesel production. The high-throughput, low-power capabilities of theround and elliptical radially concentrating systems described hereinmake these systems attractive for production scale bioreactorprocessing. The ability to perform media switching provide an additionalbenefit to many of these processes.

In antibody production, for example, spent antibody containing mediummust be extracted from the valuable hybridoma cells, preferably withoutharm to the cells. Batch methods tend to produce higher concentrationsof antibody but toxic metabolites generated during growth andcentrifugation followed by resuspension of cells can be harmful tocells. This method also requires greater technician time and poses morecontamination risks than continuous culture methods which use membranesor permeable capillary fibers for metabolite removal and nutrientreplenishment

One embodiment of the present invention provides for acoustic mediumswitching to transfer cells directly into fresh medium at optimal cellgrowth times. If a second incubation chamber is used to begin a newculture, cultures can be continuously grown in a hybrid batch/continuousmode in which spent media is harvested while new media is seeded atoptimal cell density. Excess cells can easily be removed from confluentcultures and dead cells can be removed acoustically as their acousticcontrast is lower. A simple light scatter detector can also beincorporated into the acoustic concentration device to monitor cellgrowth (see FIG. 18).

FIG. 18 illustrates a schematic example of a two chamberculturing/harvesting vessel using acoustic washing to harvest spentmedia and place cells in fresh media according to one embodiment of thepresent invention. The optical detector is used to non-invasivelymonitor cell growth at any time. Cells are cultured in chamber 1801 andperiodically sent to be acoustically focused in the switching channel1805. There they are examined for cell density/growth by the opticaldetector 1807. When growth and product production goals are met and themedia is spent cells are sent through the channel 1805 and valves areactivated to allow fresh media from the reservoir 1803 to be flowedalong the axis of the channel and spent media to be harvested in achamber 1811. The cells are focused into the fresh medium andtransferred to the second culture chamber 1809 were the process can berepeated in reverse such that cells are cultured in the chamber 1809 andtransferred into fresh media in the chamber 1801.

Any process requiring separation of viable cells to be recycled can besimilarly implemented. Examples include but are not limited to cells,bacteria or yeast producing other secreted proteins, biofuel producingcultures such as ethanol and butanol producing yeast or bacteria. Theacoustic separation process has been shown to be gentle on culturedcells and it enables automated continuously producing closed systems.

The high-throughput capabilities of round or oblate systems can also beput to good use in the harvesting of cells. For example, oil producingmicro algae can be readily concentrated many fold at high flow rateswith the process being readily scaled up by using multiple capillaries.The relatively large size of micro algae also permit high throughput forlarger diameter, lower frequency capillaries.

Example 16 Bead Based Affinity Purifications

The utility of acoustic medium switching is not just limited to cellculture. Beads with selective coatings for the product of interest canalso be envisioned in which for example the antibody is bound to proteinA or G in an immunoreaction and is washed acoustically into a cleanbuffer where it can be removed and concentrated into the final product.This process can again be used for separation in further processes suchas biotinylation or fluorescent conjugation.

The system can be further extended for use in ligand library selection(e.g. aptamer or phage selection). The process also benefits fromacoustically washing into a high salt or modified pH core where bead toligand (e.g. aptamer or phage) affinity can be controlled to select thehighest affinity ligands from a library. In this system the method ofmultiplexed fluorescent sorting can be applied to greatly increase thenumber of targets tested in a library, thereby saving time and utilizingexpensive libraries to their fullest (see FIG. 19).

FIG. 19 illustrates a diagram of an aptamer selection from a library.FIG. 19A illustrates multiplexed beads or cells 1903 with targetmolecules 1905 incubated with aptamer library 1901. FIG. 19B illustratesin-line acoustic medium switching used to separate beads/cells 1903 fromunbound aptamers 1904. Flow is into the page. Salt and or pH of the washcore (center circle) can be adjusted to select for higher affinityaptamers. Serial washes can be performed to increase purity. FIG. 19Cillustrates beads 1903 and 1905 are sorted and the DNA/RNA 1901 bound topure populations is amplified and process is repeated with the amplifiedaptamers. Subsequent rounds would focus on individual target moleculesbut other beads or cells might still be used to identifycross-reactivity of aptamers.

In general, beads with acoustic contrast to a medium can be used as analternative to magnetic beads for virtually any purification processthat magnetic beads are used. Beads used in acoustic separation cangenerally be made cheaper than magnetic beads. Larger magnetic beadsalso tend to clump together in a magnetic field and this can trapundesired materials. While few things in biological separations aremagnetic and this gives magnetic beads a specificity advantage, the samecan be said for negative acoustic contrast beads. Medium switching canalso be accomplished for laminar flow systems with magnetic beads. Thereis utility in combining methods as well, particularly if more thanbinary separation is required. Three populations can be separated forexample, if both negative and positive acoustic contrast particles werecombined with magnetic particles.

Example 17 High Speed Valve Sorting

Acoustic medium switching of the present invention provides sorting ofin-line washed cells and particles. While an acoustic medium switchingmodule can easily be used with a conventional hydrodynamically focusedflow cytometer, this new capability is made even more powerful bysorting methods that can be implemented in a sorting acoustic cytometer.Conventional cytometer sorting can be divided into two groups ofinstruments, droplet sorters and valve sorters. Most sorting tasks areperformed by droplet sorters as they are generally much faster. Valvesorting cytometers do have advantages as they are gentler on delicatecell populations, they are less expensive and tend to operate morereliably without operator intervention. The shortcomings of conventionalvalve sorting cytometers include relatively slow sorting rates of300-500 cells per second and dilution of sample with sheath fluid.Dilution with sheath fluid is also an issue for droplet sorters, butthis is less problematic as the method allows capture of cells in verysmall droplets which can be diverted to tubes containing whatever mediumis desired. The acoustic cytometer does not require sheath, so thedilutive can be eliminated. Also, since no sheath is required, sortingcan be done in a sequential manner without further dilution of sample.For relatively rare cells, this enables a high speed initial valve sortthat captures a cell of interest along with other cells for every sortdecision. This enriches the ratio of desired cells and lowers theoverall population in the sorted fraction. This sorted fraction can thenbe run again at a slower rate that will enable high purity of cells. Iffor example, cells are analyzed at a rate of 30,000 cells per second andthe valve sort were capable of sorting at 300 cells per second, eachinitial sort decision should contain an average of about 100 cells. Ifthese 100 cells are then transferred to a second sorter (or the samesorter after the initial sort) at a slower flow rate, the individualcell of interest can then be sorted with high purity. For an acousticcytometer this repeated sorting can be done without additional sheathdilution and can even be done in an instrument that reanalyzes andresorts in-line (see FIG. 20). Dual or multistage sorting can beaccomplished in-line because refocusing of cells or particles after thefirst sort can be accomplished using another acoustic focusingcapillary. A conventional sorter would require a second sheath whichwould greatly increase fluidic complexity while continuing to dilute thesample.

FIG. 20 illustrates an example of a dual stage acoustic valve sorter2000. This design enables in-line non-dilutive high speed sorting ofrare cell populations. While similar “pre-sorting” strategies usingrepeated serial valve sorts are executed in conventional sorters, thehydrodynamic focusing of these instruments results in serial dilution.Sample 2001 comprising particle 2007 is introduced into part 2001 ofsystem 2000. A first acoustic focusing system 2002 induces an acousticwave in channel 2004. Interrogation source 2013, for example a lightsource interrogates the sample and/or particle at an interrogation point2006. Particle of interest is sorted at 2009 and the unwanted particleis directed to waste 2012 paste valve 2010 a. The kept particle orparticles are directed or flowed in the stream to the second transducer2002 where a second acoustic wave can be induced into the channel. Theacoustically focused particle 2011 is interrogated at an interrogationpoint with a light source 2015 and optical information may be collected.A particle can be sorted to waste 2010 b or sent for analysis orcollection in the system 2019.

An alternative approach enabled by sheathless cytometry is the triggeredcapture of target cells. In this method a cell population can beanalyzed at high speed and when a cell with the correct profile isidentified, flow is stopped and the individual cell is collected.

In order for the many benefits of acoustic focusing to be fullyrealized, it is desirable for particle rates to be maximized forcompetitive throughput with conventional cytometers. This can beaccomplished by adjusting sample concentration in the acoustic cytometerby prior dilution, in-line dilution or acoustic washing or a combinationof these methods. For dilute samples, acoustic cytometry already has adistinct advantage, but this can be leveraged further by acousticpre-concentration or washing prior to analysis.

Prior Dilution

The simplest method for reducing coincident rates in a large diameteracoustically focused channel for concentrated samples is to dilute thesample prior to processing such that the optimum concentration ofparticles is presented for analysis. Diluting the sample prior toprocessing gives the user tremendous flexibility in the initialconcentration of samples that can be used with the instrument whileinsuring that the optimum rate of particle analysis for a given flowrate is achieved. Prior dilution can only be used in conventionalcytometry for very concentrated samples before decreasing throughput.The particle analysis rates in an acoustic cytometer can be kept highnot only because of the concentration effect but because systems havebeen developed that are capable of very high sample throughputs on theorder of several milliliters a minute. It is the high volumetric samplerate possible in acoustic systems that makes this method fundamentallydifferent from prior art methods.

Example 18

For a 300 micron diameter acoustic focusing capillary, a 10 microsecondtransit time through the interrogation laser, and a particle rate of10,000 particles/s, a concentration of about 2.8×10⁵ cells/ml or less isrequired to achieve a mean event rate of less than one in ten timewindows. According to Poisson statistics, this corresponds to aprobability of about 1% that a time window will contain more than oneevent meaning about 10% of events will be coincident. This sample isless than half the concentration than the example above for aconventional cytometer where particle rate was limited to 100 particlesper second. The volumetric flow rate required for this 10,000 particle/srate example is about 2.1 ml per minute. An acoustic cytometer canmaintain similar precision of focus to the slow sample rate of aconventional cytometer for cell sized particles at this greatervolumetric flow rate.

For an acoustic cytometer with a 300 micron diameter, a concentration ofabout 2.8×10⁵ cells/ml is optimal for maximum throughput with about 10%coincident events. For larger particles or larger laser beams or iffewer coincident events are desired, a user might choose to reducecoincident events by decreasing concentration. Samples run on anacoustic cytometer with a flow rate of 2.1 ml/min can be diluted up to210 fold before more time is needed to process the sample than for aconventional cytometer running with a sample rate of 10 μl/min. Thus,with simple up-front dilutions, an acoustic cytometer can operate athigher throughput than a conventional cytometer for concentrations up toabout 6×10⁷ cells/ml. For higher concentrations, throughput cannot beincreased beyond the maximum particle rate of a given instrument.

The 6×10⁶ cells per ml concentration sample can be conventionallyprocessed at a maximum rate of 1000 cells/s. An input rate ofapproximately 10 μl/min is typically diluted about 20 fold to reach theoptimum concentration for an acoustic cytometer. By running at 2 ml/min,particles are analyzed at nearly 10 times the rate of a conventionalcytometer. If a user prefers to take advantage of longer transit timesthrough the laser, a sample could be slowed to 0.2 ml per minute whereit would have similar particle analysis rates to the conventionalcytometer but with much longer transit times.

Prior dilution of samples with concentrations greater than an optimalconcentration for a given acoustic cytometer allows the use of nearlyany concentration of starting sample and it allows pre-treatment ofbuffers in any number of ways including adding reagents or changingacoustic contrast, dissolved gas content or temperature. It also conveysother valuable assaying benefits. Among these are background reductionfrom unbound labeled ligands and decreasing the minimum size samplerequired.

Background Reduction

When staining cells or particles with fluorescent antibodies or otherligands, it is desirable to bind as much of the ligand to the target aspossible while leaving as little as possible in solution. The remaininglabeled ligand causes fluorescent background that reduces the signal tonoise ratio during analysis.

It is usually advantageous to do the staining in a small concentratedvolume to favor binding kinetics. Simply diluting the sample reduces theconcentration of unbound antibody and therefore the background signalduring analysis. Dilution is not generally performed in conventionalcytometry because it decreases particle rate thereby increasing analysistime. For many samples in cytometry, the cell concentration is alreadygreater than a typical optimum concentration for an acoustic cytometer.

After staining, centrifugation followed by resuspension in a new bufferor medium is often performed to eliminate unbound ligands. This canstill be done for an acoustic cytometer but it can also be coupled todilution by simply adding more buffer. This process makes the unboundligand concentration even less than with centrifugation alone.

Sample Size

Conventional cytometers usually require volumes on the order of a fewhundred microliters to function properly and often some of the samplecannot be analyzed when the level runs low. Even newer cytometers withsmaller sampling capabilities boast a minimum sample volume of 10-25 μl.Using dilution, nearly any starting volume is possible in an acousticcytometer of the present invention. For example, the 210 fold dilutionfactor mentioned above makes a 1 μl sample into a 210 μl sample whichcan easily be processed with an acoustic cytometer using standard sampletubes.

One embodiment of the present invention comprises very small samples inmicrotiter plates. Well plates typically only have a maximum volume upto about 20 μl so dilution of a 1 μl sample can only be done up to 20fold. If however, diluent is fed to the well while the sample is beingfed to the cytometer, a higher fold dilution can be accomplished.

Kinetic Dilution

The rapid volumetric processing rate of an acoustic cytometer of thepresent invention also allows dynamic experiments on time scales thatconventional cytometers cannot achieve. If a diluent containing areagent(s) or drug candidate is mixed with a sample just prior toanalysis, processes triggered by this reaction can be monitored for theentire sample over a very short time period. If for example a 10 μlsample of cells with a starting concentration of 2×10⁶ cells per ml isprimed with probes for monitoring calcium activation, and 90 μl ofdiluent containing a calcium activator or test compound is added justprior to analysis, the entire sample can be analyzed in an acousticcytometer in approximately 6 to 30 seconds at a flow rate of 1 to 0.2ml/min. For the same sample in a conventional cytometer, it would takeat least 60 seconds to analyze with no dilution at the cytometer's top,less precise sample rate.

One advantage of the large dilution that is allowed in acousticcytometry of the present invention is that rapid mixing can easily beaccomplished. This ensures that all cells have equal exposure to thereagent and the cell reaction over time can be more accuratelymonitored.

Combining Pre-Dilution with In-Line Dilution

While prior dilution has many advantages, it is also sometimes desirableto combine predilution in the present invention. One example is when alarge volume of concentrated sample must be processed from a smallsample tube. A 1 ml sample with 2×10⁷ cells per ml needs to be dilutedin a conventional cytometer 100 fold to achieve a 10% coincidence rateand this requires a 100 ml sample volume. With an acoustic cytometer, itis possible to drastically change the in-line dilution from nothing toratios of diluents to sample similar to conventional hydrodynamicfocusing without degrading the precise focus.

A variable diluent can be employed with pre dilution such that virtuallyany combination of acceptable coincidence is achieved. For the exampleabove, if a 10 μl sample tube were available, 10 fold predilution couldbe combined with 10 fold in-line dilution, or 5 fold pre-dilution mightbe employed with 20 fold in-line dilution to accomplish the same thing.

The sample inputs can be configured in a number of ways for in-linedilution. If the sample flows in the center of the flow cell foracoustic focusing, and the diluents surround it coaxially, some of thebenefit of unbound probe dilution will be lost but the in-line diluentswill keep the sample from contacting the walls and will also forceparticles into starting positions in the flowed where the acousticgradient is higher. This allows greater throughput and better focusingof smaller particles.

In-line acoustic washing of particles can also be employed to the sameeffect if the sample fluid is of lesser acoustic contrast than thediluent's fluid. In this case, depending on the initial flowconfiguration, the sample fluid itself moves toward or is maintained atthe walls of the focuser, while the particles or cells are retained ator are moved to the central focus.

Combining Analysis with Off-Line Acoustic Washing/Concentration

In another embodiment of the present invention, analysis can be doneafter acoustic washing and/or acoustic concentration is performedoffline. In an acoustic washer/concentrator cells or particles can beconcentrated many fold while discarding most (concentration) or nearlyall (washing) of the original medium. This is of course of particularutility when the original concentration is sub-optimal for the desiredparticle analysis rate, but it is also of great utility for samples withvery high background or for samples that require a high degree ofbackground reduction.

Once a sample is washed or concentrated, it can then be diluted or notdepending on concentration and desired particle coincidence vs. analysisrate. It is often difficult or impractical to keep careful track of theprecise concentration of a sample so a user may employ an aid such as aspectrometer to determine concentration based on light scatter.Alternatively, another embodiment of the present invention includes anon-board spectrometer that can calculate the proper dilution andpossibly also execute the dilution automatically. Still anotherembodiment of the present invention, allows a user to take a portion ofthe sample or a diluted portion and run it on the instrument todetermine concentration and dilution prior to the main analysis.

Transit Time Advantage

For a conventional cytometer, transit times through an interrogationlaser are usually about 1-6 microseconds. With an average event rate of0.1 per unit time, 10 microseconds corresponds to an analysis rate of10,000 particles per second. For acceptable coincidence and an eventrate of 1000 particles per second, an acoustic system of the presentinvention can accommodate transit times of 100 microseconds, a rangethat greatly improves photon statistics and opens the field ofapplication for the longer acting photo-probes.

Assaying for cells, particles and microbes can be improved usingacoustic focusing with the pre-dilution method, in-line dilution methodor in-line or offline acoustic concentration or washing method orcombinations thereof. Both assaying with higher sensitivity/resolutionand novel assaying made practical by acoustic cytometry greatly expandthe capability of analysis in flow.

General examples of assaying that can use acoustic cytometry accordingto embodiments of the present invention include, but are not limited tocell sorting, apoptosis analysis, cell cycle studies, fluorescentprotein detection, cell proliferation assaying, immunophenotyping,antigen or ligand density measurement, gene expression or transfectionassaying, viability and cytotoxicity assaying, DNA/RNA content analysis,multi-plex bead analysis, stem cell analysis, nuclear stainingdetection, enzyme activity assaying, drug uptake and efflux assaying,chromosome analysis, membrane potential analysis, metabolic studies andreticulocyte and platelet analysis among others. Assaying can beimproved using acoustic focusing fluid reorientation or a combinationthereof using an acoustic cytometer with the additional steps ofadjusting to the desired optimal throughput concentration through priordilution, in-line dilution and or acoustic washing and selecting theappropriate transit time for best results. In addition, an acousticcytometer that has slow or stopped flow imaging capabilities providesadditional flexibility and advantage. Additionally, off-lineconcentration can improve throughput where cell concentrations are suboptimal. Off-line acoustic washing can also replace most centrifugationsteps or can be added as a background reducing step.

Non-Compensation Protocols

An embodiment of the present invention comprises a method for reducingcompensation in an acoustic cytometer. This embodiment includes flowingparticles with at least 2 fluorescent labels through the acousticcytometer and collecting fluorescent signals from the particles as theypass an interrogation point. Then overlap from different colorfluorescent labels is reduced by using at least one fluorescent bandfilter with a narrowed band pass such that signal from at least onefluorescent label emission is reduced. The transit time is then slowedby reducing the flow rate such that at least as many photons arecollected from the reduced signal as when the wider band pass filter isused with a faster transit time. Assaying of this embodiment preferablyuses at least 2 fluorescent labels and can run without runningcompensation controls and without compromising results.

Analysis of Microbes

Analysis of very small particles and cells is a considerable challengefor conventional cytometry. Quantities of proteins and DNA are on theorder of 3 orders of magnitude smaller then for organisms such asbacteria. The longer transit times in acoustic cytometry improve microbeanalyses by improving the photon statistics of these dim measurements.This improvement provides for many new methods of microbe analysis in anacoustic cytometer that could not commonly be measured in conventionalcytometers.

Reductions in instrument cost that are made possible by acousticcytometers also make routine counting and live/dead type analyses ofmicrobes much more accessible to more researchers.

Low cost analysis for mammalian cells may be done with the presentinvention, beyond counting and viability such as apoptosis and cellproliferation.

Methods for Increasing Dynamic Range

Increasing dynamic range can be important for assaying in which there isa wide range of signal intensity, there are increasing numbers ofdistinguishable populations in a bead set and using detectors that havea more limited dynamic range. Photo-multiplier tubes are dominant incytometry. They have a wide dynamic range but lower quantum efficiencythan some lower dynamic range detectors including but not limited toavalanche photodiodes (APDs). Multi-pixel APD devices known as siliconPMTs may also be used in the present invention.

With the long transit times in an acoustic cytometer, greater dynamicrange is available than in fast transit time systems because of theadded dimension of time. Two lasers of different power and differentspatial location may be used to analyze the same particle twice. Thestronger laser is used to quantify the dim particles while the weakerlaser is used for stronger particles. For a longer transit system, asimilar increase in dynamic range can be realized by using a singleweaker laser, increasing transit time and measuring pulse area(integrated signal from photons). Alternatively, instead of, or inaddition to, measuring peak height, the rate of signal increase ordecrease of the pulse can be measured. For a large signal, if thisinformation is taken prior to detector saturation, the expectedbrightness can be calculated rather than measured. This method isparticularly useful for beadsets with set ratios of coding labels as theratio(s) of rate increase can be used to decode the population withoutregard to intensity. Also, an extremely wide range of stainingconcentrations can be used without saturation of the detector.

The longer transit times also make increased dynamic range from a singlemodulated or pulsed system more practical. In a modulated system, dimparticles are measured at peaks and bright particles are measured atvalleys. To use a single laser for a pulsed system both stronger andweaker pulses need to be administered at different times. For a pulserest method, this is practical for long transit times but not shorttransit times where the rest period is a significant fraction of thetransit time. In a pulsed system, peak intensities can be very high andcan damage certain types of photodetectors, so care must be taken inphotodetector selection.

Still another method for increasing dynamic range is decreasing thecolor bandwidth of filters. The most common example of this is a lineardetector array in a spectrometer used in conjunction with a dispersiveelement including but not limited to a grating or prism. While thislimits the number of photons per detector and therefore decreasesprecision due to photoelectron statistics, it allows brighter signalswithout saturation and can also be used to reduce compensationrequirements in multi-color assaying and reduce signal to noise bycollecting a higher ratio of signal light to background light. If forexample one constructed assaying using AlexaFluor® 405 and Qdots®: 545,585, 655 and 800, there would be some spectral overlap of fluorophoresbut uncompensated detection can be used if some narrow band pass filterswere to be used.

Multi-Parameter Detection

Acoustic cytometry according to systems and methods of the presentinvention not only adds to dynamic range but it can add dimensions toassaying multiplexing by allowing enough time for other opticalphenomena to be monitored, including but not limited to luminescenceand/or chemi/bio/electrical luminescence. With the previous example, ifa metal ligand complex including but not limited to europium chelate isa sixth label and a pulsed light source, the first five colors can bemonitored just after the pulse and the Europium can be measuredthroughout its decay lifetime of several hundred microseconds. Thenarrow primary emission of the europium at 613 nm overlaps some with theemission spectra of the 585 and 655 Qdots® but it would not be detectedin these channels if a narrow emission bandpass filter is applied to theQdot channels. With this combination, six colors are possible withvirtually no compensation. In general, long emission fluors can beautomatically compensated for even if there is bleed over becauserelative contributions can be determined on the basis of time and can besubtracted appropriately. Given the complexity of controls and computingpower required in typical six color flow assaying, an embodiment of thepresent invention provides for compensation free or minimal compensationreagent kits, even down to two colors.

Assaying is often processed in a single sample, multi-parameterdetection can have great utility. Short lived fluorescence intensitybeads can be used as an assaying identifier and long lived fluorescencelifetime as a reporter. With longer transit times and optimizedthroughput, there are many useful applications. If, for example severalshorter lived probes are incorporated into a beadset with varyingintensities, the number of possible combinations is such that thebeadset can compete with conventional high density nucleic acid arrays.With luminescent reporters, very high sensitivity is possible even withhighly fluorescent beads. Additionally, the combination of Qdots® andmetal ligand complexes can be efficiently excited with a single violetsource including but not limited to a 375 nm laser diode.

Autofluorescence Correction

Auto-fluorescence is often a problem for sensitive detection of smallnumbers of labels. It has fairly broad emission and can spill over intomany channels. In multi-colored applications, it adds another parameterthat must be compensated outside of the multiple labels to be used. Justas longer transit times can help improve coefficients of variation forlabels with better photoelectron statistics it can also help reducevariance from background such as auto-fluorescence. The net result isthat signal to noise ratio is improved as the variance of both signaland background is narrowed.

Auto-fluorescence subtraction has been demonstrated using two lasers.The first laser excites auto-fluorescence above the wavelength of theexcitation laser, and the signal detected above that wavelength is usedto estimate the auto-fluorescence contribution expected for the primarydetection laser. Auto-fluorescence can be done with a system having aviolet laser and a blue laser. It can also be done with a system thathas only a violet laser or is using a violet laser to excite more thanone color, if there is a separate color band to monitor theauto-fluorescence. Only the blue fluorescence channel is monitored, andexpected contribution in other channels is subtracted. For pulsed ormodulated systems with long lifetime probes, the short livedcontribution of the auto-fluorescence combined with the initial outputof the long lifetime probe is measured. Fluorescence of the longlifetime probe after the auto-fluorescence has decayed is also measuredand back calculated to determine the auto-fluorescence contribution inall channels.

Example 19

Four color assaying with only auto-fluorescence compensation can beperformed using four different Qdots®: 525, 585, 655 and 800 and asingle violet diode laser. These Qdots® have very little spectraloverlap and can be easily separated. If a second laser, including butnot limited to an inexpensive diode such as 650 nm or 780 nm is added,other combinations that are virtually compensation free can be addedwith even more colors. For example, Qdots® 525, 565, 605, 705 andAlexaFluor750 which is excited very efficiently at 780 nm can be added.The 800 Qdot® is not chosen in this case as it has some excitation at780 nm. For this five color combination, narrow band filters are used toprevent overlap between Qdots®. If elimination of compensation is notcritical, similar strategies for employing low cost diodes can be usedeffectively with more conventional dye combinations such as pacific blueAlexaFluor405®/Cascade Blue® and pacific Orange® off the violet diodeand APC and APC AlexaFluo®700 off a 650 nm diode. Alternatively, 473 nmDPSS blue lasers are reasonably inexpensive when they have RMS noiselevels of a few percent or more. The long transit times afforded by anacoustic cytometer enable noise integration that can make these lasersattractive. These lasers can then be used in place of the most common488 nm wavelength lasers where they are capable of exciting the mostcommon fluorophores. Green DPSS modules e.g. (532 m) are even lessexpensive and less noisy and can be used to excite PE and its conjugatesmore effectively than even the 488 nm wavelength. In a system whereemissions off of each laser are kept distinct, either by spatial ortemporal separation, one can use several colors from each laser. If thepulse/rest method is used, lasers can be co-located and fired insequence. Fluorophores that have little absorption in bands that arebeing pulsed are still able to rest. If, for example, the rest period isone microsecond and four different lasers are used with 10 ns pulses,each laser is triggered every microsecond with a pulse of a differentwavelength hitting the target every 250 ns. A second low power pulse foreach laser can be used to extend dynamic range (brightest signals arequantified from the low power pulse, dimmest from the high power pulse).Using lasers at 405 nm, 532 nm, 650 nm and 780 nm four colors andautofluorescence can be monitored with virtually no compensation: 405nm-autofluorescence and Pacific Orange, 532 nm-PE or Cy®3, 635nm-AlexaFluor®647 and 780 nm-AlexaFluor®790. There is some excitation ofPE at 405 nm and some excitation of AlexaFluor®790 at 635 nm so slightcompensation might be required. If compensation need not be eliminated,several colors can be excited off of each laser. With lasers collocatedbut separated temporally, one can use the same detectors where dyeemissions from fluorophores excited by different lasers overlap.

Many of the 405 nm violet laser diodes are typically high quality withlow noise. Since these diodes can be obtained inexpensively with highpulsed powers, they useful for implementing high power pulses with longrest times. The diode wavelength of the 405 nm violet laser can be veryuseful with or without pulses when coupled with long transit times. Itis very efficient for excitation of quantum dots which is useful formany-colored assaying. This, coupled with narrow band emission filters,is useful for assaying with little or no need to compensate.

Example 20 Method for Luciferase Mediated Gene Detection in an AcousticCytometer

Another embodiment of the present invention provides form bio orchemiluminescence in an acoustic cytometer and the detection of geneexpression, for example, using luciferase as a gene reporter. While geneexpression detection can be accomplished with other means such asfluorescent protein expression, bio/chemi luminescence adds anadditional parameter that can be separated in time from this or otherflow cytometry fluorescence parameters. Light generated by the reactionof gene expressed luciferase and its substrates luciferin (orcoelenterazine for Renilla luciferase) does not require externalexcitation and is therefore free of autofluorescence excited in thecell, flow cell or detection optics. This also makes luciferaseespecially useful for detection of low level gene expression wheresignal to noise is especially important. In general, cells expressingluciferase are loaded with luciferin which is generally cell impermeantexcept for specialized reagents such as caged DMNPE luciferin which canbe loaded into the cell by incubation. They are then supplied ATP whichcompletes the light producing reaction. For DMNPE luciferin can beuncaged using UV light.

Using an acoustic cytometer with a pulsed excitation system, it ispossible to sensitively monitor the chemi-luminescence between laserpulses. Standard fluorescence flow cytometry parameters can be collectedas desired to determine cell characteristics including cell surfacemarkers, detection of fluorescent protein gene expression, calciumactivation, nucleic acid analysis and so forth.

Luciferase Antibodies and Secondary Reagents

Luciferase can also be conjugated to antibodies and secondary reagentslike protein A and G. Avidin and streptavidin recombinant protein A andstreptavidin luciferase fusion proteins have also been developed. Thesereagents can be used to label cellular antigens or bead bound targets inorder to add an additional parameter for analysis in acousticcytometers. With an acoustic wash containing luciferin and ATP, systemswith pulsed lasers can detect the luminescence between pulses andsubtract this quantity of light from overlapping spectra of fluorophoresused to measure other targets. This is especially useful for multiplexbeads sets that rely on fluorescence for coding. It is also especiallyuseful for measuring low levels of antigens on cells with highautofluorescent background. In addition, luciferase can be used inconjunction with any laser combination or even in the absence of lasersas it does not require excitation light.

Materials for Engineered Acoustic Media

According to another embodiment, methods of acoustic washing particlesand reorienting fluid utilize media formulations that have higheracoustic contrast than the sample medium. For many biological samplesthe medium is buffered saline, often with protein, detergents or otheradditives. Many media with higher acoustic contrast than physiologicalsaline have been developed for use in density gradient separations bycentrifugation. The functional constituents of these media are salts andproteins combined with additives used to increase specific gravitywithout undue increase in salinity. Commonly used examples includesucrose, polysucrose, polydextran, glycerol, iodinated compounds likeamidotrizoate, diatrizoate, iohexol (Nycodenz®), iodixanol, ioxaglate,iopamidol, metrizoate, metrizamide, and nanoparticulate material such aspolymer coated silica (Percoll® for example). In applications where highsalinity is not a problem, the primary constituent is a heavy salt suchas cesium chloride or potassium bromide.

For acoustic separations density and osmolarity are important butadditional parameters such as compressibility and viscosity are moreimportant than for centrifugation media. This makes the priorities forformulation of acoustic separation media different. Viscosity is ofhigher concern than for centrifugation as higher viscosity dissipatesmore acoustic energy relative to lower viscosity. Therefore, compoundsthat contribute to high viscosity are not preferred unless required bythe application. In general sucrose/polysucrose, glycerol and dextranfit into this category. Nano silica coated with polyvinylpyrrolidone isalso highly viscous and fluorescent as well. Preferable compounds to beadded include the iodinated compounds above and are preferably selectednot only on the basis of contribution to viscosity and osmolarity butalso on compressibility. Metrizamide, Nycodenz®, diatrizoate andiodixanol are useful for altering the acoustic contrast of a fluid.

In preparations where physiological osmolarity is desired but short termloss of physiological ions will not be important, a heavy salt, such ascesium chloride but not limited thereto, can be substituted or partiallysubstituted for other salts such as sodium chloride. Cesium chlorideprovides a benefit not only because it is an innocuous and relativelyheavy but because it reduces viscosity of the medium. A cesium chloridesolution with physiological osmolarity has about 3% lower viscosity thana comparable sodium chloride solution. This is an advantage for acousticseparations according to one embodiment where higher viscosity absorbsmore acoustic energy.

Cesium chloride is useful for acoustic separations that can toleratehigh salt such as separations of fixed cells and beads. High saltacoustic wash buffer combined with additives such as protein andsurfactant or detergent can be used to minimize nonspecific binding inboth protein and nucleic acid assaying. One preferred embodiment usescesium chloride and a Pluronic® non-ionic surfactant such asPluronic®F68. The Pluronic® has very low auto-fluoresence and istherefore well suited to flow analysis.

Beads with Lifetime Auto-Calibration

A difficult problem for assaying in flow cytometry is absolutequantification of analytes from instrument to instrument and day to dayor even minute to minute. Differences in laser power and fluctuation,PMT adjustments and degradation and flow alignment are among the worstculprits in variability. Absolute quantification must typically be doneusing calibration beads that excite and emit in the same channels as theanalyte to be detected. An alternative to this procedure can beaccomplished in an acoustic cytometer with lifetime discriminationcapability using beads loaded with a known amount of long lifetimefluorescent dye (preferably greater than 1 microsecond). The longlifetime dye is excited simultaneously with the analyte probe and aftershort-lived fluorescence dies down (typically 1-100 μs), the remainingsignal of the long lifetime probe can be used to calculate the signal ofthe analyte probe. The initial signal of the long lifetime probe iscalculated from the known lifetime curve of the dye and is subtractedfrom the combined fluorescence peak of the analyte probe and the longlifetime dye. Alternatively, if the analyte is probed with a longlifetime dye the bead reference dye can be short lived. Absolutecalibration is easiest when the analyte and reference dye are excited bythe same laser and detected by the same detector so the probes need tobe selected with this in mind. The commonly used lanthanide chelates aregenerally UV excited so their utility is limited in systems with visiblelasers. Other suitable candidates include but are not limited to metalligand complexes using metal ions such as europium, terbium, samarium,iridium, ruthenium, neodymium, ytterbium, erbium, dysprosium, platinum,palladium, and gadolinium. The excitation, emission and lifetimeproperties of metal ligand complexes are dictated by the metal ion andits ligand. Coupling different ligands to different ions and/ormodifying ligand structure has been and continues to be heavilyresearched. A wide variety of possibilities are available for tuning oflifetime and excitation and emission wavelengths.

In one embodiment of the present invention, reference beads can beformulated for any of the systems described previously by combiningcompatible optical parameters. For example absorptive dyes can be usedfor coding while a short-lived fluorescent dye is either used forreference or detection and a long-lived dye is used for reference ordetection. A UV excitable short lifetime dye including but not limitedto Pacific Orange®, or a quantum dot and long-lifetime probes includingbut not limited to terbium chelates or europium chelates or tandemsthereof, are good choices for single source excitation of the referenceand detection components. Absorptive dyes can be selected from a widelist of non-fluorescent species but they preferably absorb in spectralregions away from the reference and detection probes excitation andemission such that even very heavily dye loaded beads do not absorb theexcitation or emission light. Good choices would absorb in the infraredor near infra-red region. Low cost diode lasers in this spectral rangemake this choice even more attractive. In this spectral region it alsoworks well to use fluorescent dyes including but not limited toAlexaFlour®647 and AlexaFluor®790 for their absorption properties onlysince the emission wavelengths do not interfere with the coding anddetection regions.

Another use is quantum dots as coding labels and terbium or europiumchelates for detection with either one of the quantum dots as areference or another organic UV excitable dye as reference. In this casethe AlexaFluor®405, for example, can also function in the detector role.Qdot®545 can be used in conjunction with terbium chelate and Qdot®625can be used in conjunction with europium chelate. Many combinations areuseful with a single violet excitation source.

Still another example of reference beads that can be formulated for anyof the systems described previously uses Ruthenium ligand complexes forthe reference and a common fluorophore for the reporter such asPacificBlue® or PacificOrange® or Qdots® with violet excitation and orfluorescein/AlexaFluor®488, PE/PE conjugates or PerCP/PerCP conjugateswith violet or blue excitation. The ruthenium ligand reference of thisembodiment of the present invention has relatively broad band emissionand is well excited by both violet and blue lasers. It can therefore bemonitored in the same channels as many common fluorophores. Usingdifferent colored analyte reporters simultaneously is also possible andpermits either simultaneously monitoring more than one analyte on onebead or increasing the size of the multiplex array by using differentlytagged reporters for different analytes. A 20 analyte array can be madefor example using a single coding color (e.g. Qdot®800) of 10 differentintensities if 2 reporters are used (e.g. PacificBlue® and PE). Thesingle color array can be expanded to 40 elements if for example 2colors of reporters are monitored from each laser.

As DNA content in cells in resting phase can be very consistent,antigenic markers on cells can also be quantified relative to afluorescent DNA stain by using pulsed excitation and measuring theoverlap in signal over time with long-lifetime probes used to stain theantigenic markers. Effects not related to excitation that might causevariation in the DNA stain fluorescence relative to fluorescence of theantigenic probes should be minimized. These include temperature, pH anddye loading effects.

Referencing to a DNA stain can similarly be done with long-lifetime DNAprobes and short lifetime antigenic probes given an appropriate DNAstain.

Lifetime coding can also be combined with lifetime reference if theemission colors or the excitation of the long-lifetime elements can bewell separated. If for example, terbium chelate and a short lifetime UVexcited dye are used for coding and ruthenium is used for reference, UVexcitation light can be used for coding while violet and or blue lightis used for reference and analyte detection.

One embodiment of the present invention comprises a method forquantifying an amount of analyte bound to a particle in an acousticparticle analyzer. This method preferably includes manufacturing aparticle having a known amount of calibration dye with a long lifetimeand a specificity for an analyte. The analyte is bound to the particlesand passes the particle through an interrogation zone with a pulsed ormodulated laser. The short-lifetime fluorescent signal which relates tothe binding event in the interrogation zone is measured and theoverlapping fluorescent signal is measured from the long lifetimereference probe. The amount of analyte is then preferably calculated bycomparing the analyte related signal to the signal from the known amountof reference label. The analyte related signal is preferably generatedby binding a fluorescent ligand specific for the analyte to the analytesuch that the particle and analyte and fluorescent ligand form acomplex.

Another embodiment of the present invention comprises a method forquantifying the amount of analyte bound to a particle in an acousticparticle analyzer. In this embodiment, a particle is manufactured havinga known amount of calibration dye with a short lifetime and aspecificity for an analyte. This analyte is bound to the particle andpasses the particle through an interrogation zone with a pulsed ormodulated laser. A long lifetime fluorescent signal that is related tothe binding event in the interrogation zone is measured and theoverlapping fluorescent signal from the short lifetime reference probeis also measured. The amount of analyte is then calculated by comparingthe analyte related signal to the signal from the known amount ofreference label. The analyte related signal of this embodiment ispreferably generated by binding a fluorescent ligand specific for theanalyte to the analyte such that the particle and analyte andfluorescent ligand form a complex.

ENVIRONMENTAL AND INDUSTRIAL APPLICATIONS

In one embodiment of the present invention, acoustic concentration andwashing can be used for sample treatment and analysis in a range ofenvironmental and industrial samples, particularly where particles ofinterest are rare and require significant concentration to acquire astatistically meaningful population. The ability of acousticconcentrators to function as “filterless filters” that are not subjectto clogging and periodic replacement requirements makes them veryattractive in many applications. Analysis of microbes from municipalwater supplies is a prime example. Specific nucleic acid probes andother microbe specific probes are used to confirm the presence ofmicrobes in water samples but pre-concentration before staining isnecessary to limit the amount of staining reagent and to process enoughvolume to be statistically significant. Similar microbial testing isdone for a multitude of industrial products and foods from juice, milkand beer to mouthwash and these analyses can also benefit tremendouslyfrom acoustic concentration. Acoustic washing can be employed toseparate environmental and industrial analytes from reagents such as thestaining probes for more sensitive measurements and can also be used toreplace the original sample medium with fluids containing differentreagents or compositions. Acoustic washing using electrolyte buffer forimpedance analysis is of particular utility for virtually any sampleincluding those listed above which does not have the requiredconductivity for analysis. In an acoustically focused imager, analysiscan extend to shape and size of particles which is important for a greatdeal of industrial processes as diverse as ink production for copiersand printers and quality control in chocolate making. Acoustic focusingand alignment of particles greatly enhances quality of imaging ofparticles by bringing particles into focus at the focal imaging planeand also orienting asymmetric particles with respect to the acousticfield. Acoustic focusing can be used to concentrate and/or removeparticles from waste streams or feed streams. An acoustic focusingapparatus can be placed in with other filtration systems, e.g. waterpurification systems, to extend the life of the filters. Such processingis not just limited to aqueous environments, removal of metal, ceramicor other particulates from machining fluids or particulates from spentoils such as motor oils and cooking oils is also possible.

Any of the methods above can be automated with a processor and adatabase. A computer readable medium containing instructions canpreferably cause a program in a data processing medium (a computingsystem) to perform a method.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described components and/oroperating conditions of this invention for those used in the precedingexamples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above are hereby incorporated by reference.

What is claimed is:
 1. A method for acoustically manipulating a particleusing radial acoustic focusing comprising: introducing into a channel afirst fluid having a first acoustic contrast with a population ofparticles suspended therein; introducing into the channel a second fluidhaving a second acoustic contrast that is greater than the firstacoustic contrast of the first fluid; flowing the first and second fluidin the channel; applying a radial acoustic radiation pressure to thechannel; and acoustically focusing at least a portion of the populationof particles from the first fluid to the second fluid, wherein theportion of the population of particles are acoustically focused relativeto the first acoustic contrast of the first fluid and the secondacoustic contrast of the second fluid, and wherein the portion of thepopulation of particles in the second fluid is acoustically focused intoa single file line to pass through an interrogation zone.
 2. The methodof claim 1, wherein acoustically focusing at least the portion of thepopulation of particles is for assaying the particles or fluids toproduce an assay.
 3. The method of claim 2, wherein the first fluid is abiological fluid selected from the group consisting of blood, bonemarrow, semen, vaginal fluid, urine, spinal fluid, sputum, bile,peritoneal fluid, and aspirate from hollow organs, cysts and tissue. 4.The method of claim 1 further comprising: producing an acoustic nodeoutside the channel; and wherein the second fluid with the at least aportion of the particles therein is directed to the top surface of thechannel near the acoustic node.
 5. The method of claim 1 furthercomprising moving the first fluid in a first laminar flow stream andmoving the second fluid in a second laminar flow stream.
 6. The methodof claim 1 wherein the population of particles comprises a subset ofparticles having a first particle size and a second particle sizewherein the first particle size is greater than the second particlesize.
 7. The method of claim 6 wherein the first particle size isacoustically focused more quickly into the second fluid than the subsetof particles having a second particle size.
 8. The method of claim 7further comprising collecting the population of particles by size. 9.The method of claim 8, wherein the collected particles have a differentcoefficient of variation as compared to the population of particlesbefore the introducing into the channel.
 10. The method of claim 1wherein the first fluid comprises a reagent that specifically binds atleast a subset of the portion of the population of particles.
 11. Themethod of claim 10 wherein the reagent binds to at least a portion ofthe subset of the population of particles to form a particle-reagentcomplex having an acoustic contrast different than the acoustic contrastof the portion of the population of particles not complexed withreagent.
 12. The method of claim 11 wherein the particle-reagent complexis acoustically focused away from at least a portion of the populationof particles not complexed with reagent.
 13. The method of claim 10wherein the step of acoustically focusing at least a subset of thepopulation of particles from the first fluid to the second fluidcomprises acoustically focusing the subset away from reagents in thefirst fluid.
 14. The method of claim 13 further comprising collectingthe subset from the second fluid.
 15. The method of claim 1 wherein thestep of acoustically focusing comprises concentrating the population ofparticles into the second stream.
 16. The method of claim 1 furthercomprising analyzing in a particle analyzer the portion of thepopulation of particles in the second fluid.
 17. The method of claim 16wherein the particle analyzer is a flow cytometer.
 18. The method ofclaim 1 further comprising imaging with an imager the portion of thepopulation of particles in the second fluid.
 19. The method of claim 1further comprising non-dilutive sorting of the portion of the populationof particles in the second fluid.
 20. The method of claim 1 wherein thesecond fluid comprises a reagent that reacts with at least some of theparticles from the portion of the population of particles acousticallyfocused into the second fluid.
 21. The method of claim 20 wherein thepopulation of particles comprises an array of beads having a targetspecific for a pre-bound fluorescent analyte of interest and wherein thesecond fluid is suspected of containing non fluorescent analyte that iscapable of binding specifically to its target on a bead from the arrayof beads and further comprising: displacing the pre-bound fluorescentanalyte with a non-fluorescent analyte when the bead is acousticallyfocused in the second fluid; and analyzing the array of beads forfluorescence.
 22. The method of claim 20 wherein the reagent isantibodies or aptamers specific for particle antigens.
 23. The method ofclaim 20 wherein the reagent is ligands specific for particle receptors.24. The method of claim 20 wherein the reagent is enzyme specific forparticle substrate.
 25. The method of claim 20 wherein the reagent isstains specific for particle nucleic acid.
 26. The method of claim 20wherein the reagent is an antigen specific for a particle antibody. 27.The method of claim 20 wherein the reagent is an analyte specific for aparticle target.
 28. The method of claim 20 wherein the reagent is asecondary reagent specific for one or more of a) antibodies and aptamersspecific for particle antigens; b) ligands specific for particlereceptors; c) Enzyme specific for particle substrate; d) stains specificfor particle nucleic acid; e) antigens specific for particle antibodies;f) an analyte specific for a particle target.
 29. The method of claim 1wherein the first fluid comprises a reagent that reacts with at leastsome of the population of particles.
 30. The method of claim 29 whereinthe reagent is antibodies and aptamers specific for particle antigens.31. The method of claim 29 wherein the reagent is ligands specific forparticle receptors.
 32. The method of claim 29 wherein the reagent isenzyme specific for particle substrate.
 33. The method of claim 29wherein the reagent is stains specific for particle nucleic acid. 34.The method of claim 29 wherein the reagent is antigens specific forparticle antibodies.
 35. The method of claim 29 wherein the reagent isan analyte specific for a particle target.
 36. The method of claim 29wherein the reagent is secondary reagents specific for one or more of a)antibodies and aptamers specific for particle antigens; b) ligandsspecific for particle receptors; c) Enzyme specific for particlesubstrate; d) stains specific for particle nucleic acid; e) antigensspecific for particle antibodies; and f) an analyte specific for aparticle target.
 37. The method of claim 1 wherein the second fluid islocated at a center of the channel.
 38. The method of claim 1 whereinthe population of particles is selected from the group consisting ofcells, liposomes, beads and any combination thereof.
 39. The method ofclaim 38 wherein the particle is a cell and the method furthercomprising: acoustically focusing the cell into a reagent loaded secondfluid; and providing to the cell an electric field that permeates thecell membrane to permit the reagent to cross into the permeated cell.40. The method of claim 1 further comprising flowing the particle in thefirst fluid or the second fluid to a particle analyzer that is in linewith the channel.
 41. The method of claim 1 wherein the population ofparticles is cultured cells, the first fluid is cell growth medium inwhich the cells are grown and the second fluid is new cell growthmedium.
 42. The method of claim 1 wherein the population of particles ismicrosphere beads.
 43. The method of claim 1 wherein a negative contrastparticle is within the population of particles.
 44. The method of claim43 further comprising: capturing a particle of interest from thepopulation of particles with the negative contrast particle; and forcingthe particle of interest and the negative contrast particle toward awall of the channel away from a center of the channel.
 45. The method ofclaim 1 wherein the population of particles is selected from microbes,blood cells and platelets.
 46. The method of claim 1 wherein theparticle is a cell and further comprising: introducing a calciumsensitive reagent into a cell; moving the cell through a channel;acoustically focusing the cell within the channel; exposing the cell toa reagent that may or may not induce a cellular calcium response;passing the cell through an interrogation site; and collecting a signalto determine calcium concentration in the cell.
 47. The method of claim46 wherein the cell is acoustically washed prior to collection.
 48. Themethod of claim 46 wherein the cell is diluted just prior to collection.49. The method of claim 46 further comprising adjusting a flow rate toachieve a desired time of collection after the exposure to the reagentthat may or may not induce a calcium response.
 50. The method of claim 1further comprising: passing the population of particles through a zonefor collecting of luminescence; and collecting chemi, bio or electroluminescence from the particle.
 51. The method of claim 50 wherein thepopulation of particles is focused with a radial acoustic field.
 52. Themethod of claim 50 further comprising collecting luminescence betweenexcitation pulses from a light source.
 53. The method of claim 1,further comprising: introducing into the channel a third fluid having athird acoustic contrast; flowing the third fluid in the channel; andacoustically focusing the portion of the population of particles fromthe second fluid to the third fluid, wherein the second fluid is areagent stream.